Methods, kits and compositions pertaining to PNA Molecular Beacons

ABSTRACT

This invention is directed to methods, kits and compositions pertaining to PNA Molecular Beacons. PNA Molecular Beacons comprise self-complementary arm segments and flexible linkages that promote intramolecular or intermolecular interactions. In the absence of a target sequence, PNA Molecular Beacons facilitate efficient energy transfer between the linked donor and acceptor moieties of the probe. Upon hybridization of the probe to a target sequence, there is a measurable change in at least one property of at least one donor or acceptor moiety of the probe which can be used to detect, identify or quantitate the target sequence in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/888,341 (now allowed), filed on Jun. 22, 2001, which is a division ofU.S. application Ser. No. 09/179,298 (now U.S. Pat. No. 6,355,421),filed on Oct. 26, 1998, which is a continuation-in-part of U.S.application Ser. No. 08/958,532 filed on Oct. 27, 1997 (now abandoned).

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is related to the field of probe-based nucleicacid sequence detection, analysis and quantitation. More specifically,this invention relates to novel compositions and methods pertaining toPNA Molecular Beacons.

[0004] 2. Description of the Related Art

[0005] Quenching of fluorescence signal can occur by either FluorescenceResonance Energy Transfer “FRET” (also known as non-radiative energytransfer: See: Yaron et al., Analytical Biochemistry 95: 228-235 (1979)at p. 232, col. 1, lns. 32-39) or by non-FRET interactions (also knownas radiationless energy transfer; See: Yaron et al., AnalyticalBiochemistry 95 at p. 229, col. 2, lns. 7-13). The criticaldistinguishing factor between FRET and non-FRET quenching is thatnon-FRET quenching requires short range interaction by “collision” or“contact” and therefore requires no spectral overlap between themoieties of the donor and acceptor pair (See: Yaron et al., AnalyticalBiochemistny 95 at p. 229, col. 1, lns. 22-42). Conversely, FRETquenching requires spectral overlap between the donor and acceptormoieties and the efficiency of quenching is directly proportional to thedistance between the donor and acceptor moieties of the FRET pair (See:Yaron et al., Analytical Biochemistny 95 at p. 232, col. 1, In. 46 tocol. 2, In. 29). Extensive reviews of the FRET phenomenon are describedin Clegg, R. M., Methods Enzymol., 221: 353-388 (1992) and Selvin, P.R., Methods Enzymol., 246: 300-334 (1995). Yaron et al. also suggestedthat the principles described therein might be applied to the hydrolysisof oligonucleotides (See: Yaron et al., Analytical Biochemistry 95 at p.234, col. 2, lns. 14-18).

[0006] The FRET phenomenon has been utilized for the direct detection ofnucleic acid target sequences without the requirement that labelednucleic acid hybridization probes or primers be separated from thehybridization complex prior to detection (See: Livak et al. U.S. Pat.No. 5,538,848). One method utilizing FRET to analyze Polymerase ChainReaction (PCR) amplified nucleic acid in a closed tube format iscommercially available from Perkin Elmer. The TaqMan™ assay utilizes anucleic acid hybridization probe that is labeled with a fluorescentreporter and a quencher moiety in a configuration that results inquenching of fluorescence in the intact probe. During the PCRamplification, the probe sequence specifically hybridizes to theamplified nucleic acid. When hybridized, the exonuclease activity of theTaq polymerase degrades the probe thereby eliminating the intramolecularquenching maintained by the intact probe. Because the probe is designedto hybridize specifically to the amplified nucleic acid, the increase influorescence intensity of the sample, caused by enzymatic degradation ofthe probe, can be correlated with the activity of the amplificationprocess.

[0007] Nonetheless, this method preferably requires that each of thefluorophore and quencher moieties be located on the 3′ and 5′ termini ofthe probe so that the optimal signal to noise ratio is achieved (See:Nazarenko et al., Nucl. Acids Res. 25: 2516-2521(1997) at p. 2516, col.2, lns. 27-35). However, orientation necessarily results in less thanoptimal fluorescence quenching because the fluorophore and quenchermoieties are separated in space and the transfer of energy is mostefficient when they are close. Consequently, the background emissionfrom unhybridized probe can be quite high in the TaqMan™ assay (See:Nazarenko et al., Nucl. Acids Res. 25: at p. 2516, col. 2, lns. 36-40).

[0008] The nucleic acid Molecular Beacon is another construct whichutilizes the FRET phenomenon to detect target nucleic acid sequences(See: Tyagi et al. Nature Biotechnology, 14: 303-308 (1996). A nucleicacid Molecular Beacon comprises a probing sequence embedded within twocomplementary arm sequences (See: Tyagi et al, Nature Biotechnology, 14:at p. 303, col. 1, lns. 22-30). To each termini of the probing sequenceis attached one of either a fluorophore or quencher moiety. In theabsence of the nucleic acid target, the arm sequences anneal to eachother to thereby form a loop and hairpin stem structure which brings thefluorophore and quencher together (See: Tyagi et al., NatureBiotechnology, 14: at p. 304, col. 2, lns. 14-25). When contacted withtarget nucleic acid, the complementary probing sequence and targetsequence will hybridize. Because the hairpin stem cannot coexist withthe rigid double helix that is formed upon hybridization, the resultingconformational change forces the arm sequences apart and causes thefluorophore and quencher to be separated (See: Tyagi et al. NatureBiotechnology, 14: at p. 303, col. 2, lns. 1-17). When the fluorophoreand quencher are separated, energy of the donor fluorophore does nottransfer to the acceptor moiety and the fluorescent signal is thendetectable. Since unhybridized “Molecular Beacons” are non-fluorescent,it is not necessary that any excess probe be removed from an assay.Consequently, Tyagi et al. state that Molecular Beacons can be used forthe detection of target nucleic acids in a homogeneous assay and inliving cells. (See: Tyagi et al., Nature Biotechnology, 14: at p. 303,col. 2; lns. 15-77).

[0009] The arm sequences of the disclosed nucleic acid Molecular Beaconconstructs are unrelated to the probing sequence (See: Tyagi et al.,Nature Biotechnology, 14: at p. 303, col. 1; In. 30). Because the Tyagiet al. Molecular Beacons comprise nucleic acid molecules, proper stemformation and stability is dependent upon the length of the stem, theG:C content of the arm sequences, the concentration of salt in which itis dissolved and the presence or absence of magnesium in which the probeis dissolved (See: Tyagi et al., Nature Biotchology, 14: at p. 305, col.1; lns. 1-16). Furthermore, the Tyagi et al. nucleic acid MolecularBeacons are susceptible to degradation by endonucleases andexonucleases.

[0010] Upon probe degradation, background fluorescent signal willincrease since the donor and acceptor moieties are no longer held inclose proximity. Therefore, assays utilizing enzymes known to havenuclease activity, will exhibit a continuous increase in backgroundfluorescence as the nucleic acid Molecular Beacon is degraded (See: FIG.7 in Tyagi et al: the data associated with (◯) and (□) demonstrates thatthe fluorescent background, presumably caused by probe degradation,increases with each amplification cycle.) Additionally, MolecularBeacons will also, at least partially, be degraded in living cellsbecause cells contain active nuclease activity.

[0011] The constructs described by Tyagi et al. are more broadlydescribed in WO95/13399 (hereinafter referred to as “Tyagi2 et al.”except that Tyagi2 et al. also discloses that the nucleic acid MolecularBeacon may also be bimolecular wherein they define bimolecular as beingunitary probes of the invention comprising two molecules (e.g.oligonucleotides) wherein half, or roughly half, of the targetcomplement sequence, one member of the affinity pair and one member ofthe label pair are present in each molecule (See: Tyagi2 et al., p. 8,ln. 25 to p. 9, ln. 3). However, Tyagi2 et al. specifically states thatin designing a unitary probe for use in a PCR reaction, one wouldnaturally choose a target complement sequence that is not complementaryto one of the PCR primers (See: Tyagi2 et al., p. 41, ln. 27). Assays ofthe invention include real-time and end point detection of specificsingle-stranded or double stranded products of nucleic acid synthesisreactions, provided however that if unitary probes will be subjected tomelting or other denaturation, the probes must be unimolecular (See:Tyagi2 et al., p. 37, lns. 1-9). Furthermore, Tyagi2 et al. stipulatethat although the unitary probes of the invention may be used withamplification or other nucleic acid synthesis reactions, bimolecularprobes (as defined in Tyagi2 et al.) are not suitable for use in anyreaction (e.g. PCR) wherein the affinity pair would be separated in atarget-independent manner (See: Tyagi2 et al., p. 13, lns. 9-12).Neither Tyagi et al. nor Tyagi2 et al. disclose, suggest or teachanything about PNA.

[0012] In a more recent disclosure, modified hairpin constructs whichare similar to the Tyagi et al. nucleic acid Molecular Beacons, butwhich are suitable as primers for polymerase extension, have beendisclosed (See: Nazarenko et al., Nucleic Acids Res. 25:2516-2521(1997)). A method suitable for the direct detection ofPCR-amplified DNA in a closed system is also disclosed. According to themethod, the Nazarenko et al. primer constructs are, by operation of thePCR process, incorporated into the amplification product. Incorporationinto a PCR amplified product results in a change in configuration thatseparates the donor and acceptor moieties. Consequently, increases inthe intensity of the fluorescent signal in the assay can be directlycorrelated with the amount of primer incorporated into the PCR amplifiedproduct. The authors conclude, this method is particularly well suitedto the analysis of PCR amplified nucleic acid in a closed tube format.

[0013] Because they are nucleic acids, the Nazarenko et al. primerconstructs are admittedly subject to nuclease digestion thereby causingan increase in background signal during the PCR process (See: Nazarenkoet al., Nucleic Acids Res. 25: at p. 2519, col. 1; lns. 28-39). Anadditional disadvantage of this method is that the Molecular Beacon likeprimer constructs must be linearized during amplification (See:Nazarenko et al., Nucleic Acids Res. 25: at p. 2519, col. 1, lns. 7-8).Consequently, the polymerase must read through and dissociate the stemof the hairpin modified Molecular Beacon like primer construct iffluorescent signal is to be generated. Nazarenko et al. does notsuggest, teach or disclose anything about PNA.

[0014] In still another application of FRET to target nucleic acidsequence detection, doubly labeled fluorescent oligonucleotide probeswhich have been rendered impervious to exonuclease digestion have alsobeen used to detect target nucleic acid sequences in PCR reactions andin-situ PCR (See: Mayrand, U.S. Pat. No. 5,691,146). The oligonucleotideprobes of Mayrand comprise a fluorescer (reporter) molecule attached toa first end of the oligonucleotide and a quencher molecule attached tothe opposite end of the oligonucleotide (See: Mayrand, Abstract).Mayrand suggests that the prior art teaches that the distance betweenthe fluorophore and quencher is an important feature which must beminimized and consequently the preferred spacing between the reporterand quencher moieties of a DNA probe should be 6-16 nucleotides (See:col. 7, lns. 8-24). Mayrand, however teaches that the reporter moleculeand quencher moieties are preferably located at a distance of 18nucleotides (See: col. 3, lns. 35-36) or 20 bases (See: col. 7, lns.25-46) to achieve the optimal signal to noise ratio. Consequently, bothMayrand and the prior art cited therein teach that the detectableproperties of nucleic acid probes (DNA or RNA) comprising a fluorophoreand quencher exhibit a strong dependence on probe length.

[0015] Resistance to nuclease digestion is also an important aspect ofthe invention (See: U.S. Pat. No. 5,691,146 at col. 6, lns. 42-64) andtherefore, Mayrand suggests that the 5′ end of the oligonucleotide maybe rendered impervious to nuclease digestion by including one or moremodified internucleotide linkages onto the 5′ end of the oligonucleotideprobe (See: U.S. Pat. No. 5,691,146 at col. 6, lns. 45-50). Furthermore,Mayrand suggests that a polyamide nucleic acid (PNA) or peptide can beused as a nuclease resistant linkage to thereby modify the 5′ end of theoligonucleotide probe of the invention and render it impervious tonuclease digestion (See: U.S. Pat. No 5,691,146 at col. 6, lns. 53-64).Mayrand does not however, disclose, suggest or teach that a PNA probeconstruct might be a suitable substitute for the practice of theinvention despite having obvious knowledge of its existence.Furthermore, Mayrand does not teach one of skill in the art how toprepare and/or label a PNA with the fluorescer or quencher moieties.

[0016] The efficiency of energy transfer between donor and acceptormoieties as they can be influenced by oligonucleotide length (distance)has been further examined and particularly applied to fluorescentnucleic acid sequencing applications (See: Mathies et al., U.S. Pat. No.5,707,804). Mathies et al. states that two fluorophores will be joinedby a backbone or chain where the distance between the two fluorophoresmay be varied (See: U.S. Pat. No. 5,707,804 at col. 4, lns. 1-3). Thus,the distance must be chosen to provide energy transfer from the donor tothe acceptor through the well-known Foerster mechanism (See: U.S. Pat.No. 5,707,804 at col. 4, lns. 7-9). Preferably about 3-10 nucleosidesseparate the fluorophores of a single stranded nucleic acid (See: U.S.Pat. No. 5,707,804 at col. 7, lns. 21-25). Mathies et al. does notsuggest, teach or disclose anything about PNA.

[0017] From the analysis of DNA duplexes is has been observed that: 1:the efficiency of FET (or FRET as defined herein) appears to dependsomehow on the nucleobase sequence of the oligonucleotide; 2: donorfluorescence changes in a manner which suggests that dye-DNAinteractions affect the efficiency of FET; and 3: the Forster equationdoes not quantitatively account for observed energy transfer andtherefore the length between donor and acceptor moieties attached tooligonucleotides cannot be quantitated, though it can be usedqualitatively (See: Promisel et al., Biochemistry, 29: 9261-9268 (1990).Promisel et al. suggest that non-Forster effects may account for some oftheir observed but otherwise unexplainable results (See: Promisel etal., Biochemistry, 29: at p. 9267, col. 1, ln. 43 to p. 9268, col. 1,ln. 13). The results of Promisel et al. suggest that the FRET phenomenawhen utilized in nucleic acids in not entirely predictable or wellunderstood. Promisel et al. does not suggest, teach or disclose anythingabout PNA and, in fact, the manuscript predates the invention of PNA.

[0018] The background art thus far discussed does not disclose, suggestor teach anything about PNA oligomers to which are directly attached apair of donor and acceptor moieties. In fact, the FRET phenomenon asapplied to the detection of nucleic acids, appears to be confined to thepreparation of constructs in which the portion of the probe which iscomplementary to the target nucleic acid sequence is itself comprisedsolely of nucleic acid.

[0019] FRET has also been utilized within the field of peptides. (See:Yaron et al. Analytical Biochemistry 95 at p. 232, col. 2, ln. 30 to p.234, col. 1, ln. 30). Indeed, the use of suitably labeled peptides asenzyme substrates appears to be the primary utility for peptides whichare labeled with donor and acceptor pairs (See: Zimmerman et al.,Analytical Biochemistry, 70: 258-262 (1976), Carmel et al., Eur. J.Biochem., 73: 617-625 (1977), Ng et al., Analytical Biochemistry, 183:50-56 (1989), Wang et al., Tett. Lett., 31: 6493-6496 (1990) and Meldalet al., Analytical Biochemistry, 195: 141-147 (1991). Early worksuggested that quenching efficiency of the donor and acceptor pair wasdependent on peptide length (See: Yaron et al., Analytical Biochemistry95 at p. 233, col. 2, lns. 36-40). However, the later work has suggestedthat efficient quenching was not so dependent on peptide length (See: Nget al., Analytical Biochemistry, 183: at p. 54, col. 2, ln 23 to p. 55,col. 1 ln. 12; Wang et al., Tett. Lett., 31 wherein the peptide is eightamino acids in length; and Meldal et al. Analytical Biochemistry, 195 atp. 144, col. 1, lns. 33-37). It was suggested by Ng et al. that theobserved quenching in long peptides might occur by an as yetundetermined mechanism (See: Ng et al., Analytical Biochemistry 183 atp. 55, col. 1, ln 13 to col. 2, ln 7.)

[0020] Despite its name, peptide nucleic acid (PNA) is neither apeptide, a nucleic acid nor is it even an acid. Peptide Nucleic Acid(PNA) is a non-naturally occurring polyamide (pseudopeptide) which canhybridize to nucleic acid (DNA and RNA) with sequence specificity (SeeU.S. Pat. No. 5,539,082 and Egholm et al., Nature 365: 566-568 (1993)).PNAs are synthesized by adaptation of standard peptide synthesisprocedures in a format which is now commercially available. (For ageneral review of the preparation of PNA monomers and oligomers pleasesee: Dueholm et al., New J. Chem., 21: 19-31 (1997) or Hyrup et. al.,Bioorganic & Med. Chem. 4: 5-23 (1996)). Alternatively, labeled andunlabeled PNA oligomers can be purchased (See: PerSeptive BiosystemsPromotional Literature: BioConcepts, Publication No. NL612, PracticalPNA, Review and Practical PNA, Vol. 1, Iss. 2)

[0021] Being non-naturally occurring molecules, PNAs are not known to besubstrates for the enzymes which are known to degrade peptides ornucleic acids. Therefore, PNAs should be stable in biological samples,as well as have a long shelf-life. Unlike nucleic acid hybridizationwhich is very dependent on ionic strength, the hybridization of a PNAwith a nucleic acid is fairly independent of ionic strength and isfavored at low ionic strength, conditions which strongly disfavor thehybridization of nucleic acid to nucleic acid (Egholm et al., Nature, atp. 567). The effect of ionic strength on the stability and conformationof PNA complexes has been extensively investigated (Tomac et al., J. Am.Chem. Soc. 118: 5544-5552 (1996)). Sequence discrimination is moreefficient for PNA recognizing DNA than for DNA recognizing DNA (Egholmet al., Nature, at p. 566). However, the advantages in point mutationdiscrimination with PNA probes, as compared with DNA probes, in ahybridization assay appears to be somewhat sequence dependent (Nielsenet al., Anti-Cancer Drug Design 8: 53-65, (1993)). As an additionaladvantage, PNAs hybridize to nucleic acid in both a parallel andantiparallel orientation, though the antiparallel orientation ispreferred (See: Egholm et al., Nature at p. 566).

[0022] Despite the ability to hybridize to nucleic acid in a sequencespecific manner, there are many differences between PNA probes andstandard nucleic acid probes. These differences can be convenientlybroken down into biological, structural, and physico-chemicaldifferences. As discussed in more detail below, these biological,structural, and physico-chemical differences may lead to unpredictableresults when attempting to use PNA probes in applications were nucleicacids have typically been employed. This non-equivalency of differingcompositions is often observed in the chemical arts.

[0023] With regard to biological differences, nucleic acids, arebiological materials that play a central role in the life of livingspecies as agents of genetic transmission and expression. Their in vivoproperties are fairly well understood. PNA, on the other hand isrecently developed totally artificial molecule, conceived in the mindsof chemists and made using synthetic organic chemistry. It has no knownbiological function (i.e. native (unmodified) PNA is not known to be asubstrate for any polymerase, ligase, nuclease or protease).

[0024] Structurally, PNA also differs dramatically from nucleic acid.Although both can employ common nucleobases (A, C, G, T, and U), thebackbones of these molecules are structurally diverse. The backbones ofRNA and DNA are composed of repeating phosphodiester ribose and2-deoxyribose units. In contrast, the backbones of the most common PNAsare composed on N-[2-(aminoethyl)]glycine subunits. Additionally, in PNAthe nucleobases are connected to the backbone by an additional methylenecarbonyl moiety.

[0025] PNA is not an acid and therefore contains no charged acidicgroups such as those present in DNA and RNA. Because they lack formalcharge, PNAs are generally more hydrophobic than their equivalentnucleic acid molecules. The hydrophobic character of PNA allows for thepossibility of non-specific (hydrophobic/hydrophobic interactions)interactions not observed with nucleic acids. Further, PNA is achiral,providing it with the capability of adopting structural conformationsthe equivalent of which do not exist in the RNA/DNA realm.

[0026] The unique structural features of PNA result in a polymer whichis highly organized in solution, particularly for purine rich polymers(See: Dueholm et al., New J. Chem., 21: 19-31 (1997) at p. 27, col. 2,lns. 6-30). Conversely, a single stranded nucleic acid is a random coilwhich exhibits very little secondary structure. Because PNA is highlyorganized, PNA should be more resistant to adopting alternativesecondary structures (e.g. a hairpin stem and/or loop).

[0027] The physico/chemical differences between PNA and DNA or RNA arealso substantial. PNA binds to its complementary nucleic acid morerapidly than nucleic acid probes bind to the same target sequence. Thisbehavior is believed to be, at least partially, due to the fact that PNAlacks charge on its backbone. Additionally, recent publicationsdemonstrate that the incorporation of positively charged groups intoPNAs will improve the kinetics of hybridization (See: Iyer et al., J.Biol. Chem. 270: 14712-14717 (1995)). Because it lacks charge on thebackbone, the stability of the PNA/nucleic acid complex is higher thanthat of an analogous DNA/DNA or RNA/DNA complex. In certain situations,PNA will form highly stable triple helical complexes through a processcalled “strand displacement”. No equivalent strand displacementprocesses or structures are known in the DNA/RNA world.

[0028] Recently, the “Hybridization based screening on peptide nucleicacid (PNA) oligomer arrays” has been described wherein arrays of some1000 PNA oligomers of individual sequence were synthesized on polymermembranes (See: Weiler et al., Nucl. Acids Res. 25: 2792-2799(1997)).Arrays are generally used, in a single assay, to generate affinitybinding (hybridization) information about a specific sequence or sampleto numerous probes of defined composition. Thus, PNA arrays may beuseful in diagnostic applications or for screening libraries ofcompounds for leads which might exhibit therapeutic utility. However,Weiler et al. note that the affinity and specificity of DNAhybridization to immobilized PNA oligomers depended on hybridizationconditions more than was expected. Moreover, there was a tendency towardnon-specific binding at lower ionic strength. Furthermore, certain verystrong binding mismatches were identified which could not be eliminatedby more stringent washing conditions. These unexpected results areillustrative of the lack of complete understanding of these newlydiscovered molecules (i.e. PNA).

[0029] In summary, because PNAs hybridize to nucleic acids with sequencespecificity, PNAs are useful candidates for investigation as substituteprobes when developing probe-based hybridization assays. However, PNAprobes are not the equivalent of nucleic acid probes in both structureor function. Consequently, the unique biological, structural, andphysico-chemical properties of PNA requires that experimentation beperformed to thereby examine whether PNAs are suitable in applicationswhere nucleic acid probes are commonly utilized.

SUMMARY OF THE INVENTION

[0030] Tyagi et al. and Tyagi2 et al. disclose nucleic acid MolecularBeacons which comprise a hairpin loop and stem to which energy transferdonor and acceptor moieties are linked at opposite ends of the nucleicacid polymer. Numerous PNA polymers were examined in an attempt toprepare a PNA Molecular Beacon. The applicant's have determined that allprobes they examined, which contained linked donor and acceptor moietiesexhibited a low inherent noise (background) and an increase indetectable signal upon binding of the probe to a target sequence. Verysurprisingly, these characteristic properties of a nucleic acidMolecular Beacon were observed whether or not the PNA oligomer possessedself-complementary arm segments intended to form a PNA hairpin. Forexample, PNA oligomers prepared as control samples which by design didnot possess any self-complementary arm segments suitable for forming ahairpin exhibited a signal (PNA oligomer bound to target sequence) tonoise (no target sequence present) ratio which was quite favorable ascompared with probes comprising flexible linkages and self-complementaryarm segments.

[0031] Applicant's data further demonstrates that flexible linkagesinserted within the probe and shorter self-complementary arm segmentsare a preferred embodiment since the signal to noise ratio of probes ofthis embodiment compare well with the signal to noise ratio publishedfor nucleic acid hairpins (approximately 25 to 1). The data compiled byapplicants is inconclusive with respect to whether or not the PNAMolecular Beacons they prepared which have shorter arms segments (2-5subunits in length) and one or more flexible linkages exist as hairpins.However, applicant's data demonstrates that probes with longer armsegments (e.g. 9 subunits) do form a hairpin (See: Example 19 of thisspecification) and unlabeled probes having arms segments as short as sixsubunits do not exist primarily as a hairpin (See: Example 19 of thisspecification). Furthermore, the signal to noise ratio for those probeshaving longer arm segments suitable for forming a hairpin exhibited verypoor a signal to noise ratios upon melting of the hairpin or when in thepresence of a complementary nucleic acid. Consequently, embodimentshaving longer arm segments (e.g. 6 or more subunits) do not appear to bewell suited for use in the detection of nucleic acid targets.

[0032] The data compiled by applicant's demonstrates the non-equivalenceof structure and function of PNA as compared with nucleic acids.Consequently, this invention pertains to methods, kits and compositionspertaining to PNA Molecular Beacons. Though we refer to the probes ofthis invention as PNA Molecular Beacons, we do not mean to imply thatthey exist as hairpins since they may well exist as aggregates,bimolecular constructs or as higher order hybrids (e.g. multimers).Regardless of the nature of the secondary structure, a PNA MolecularBeacon efficiently transfers energy between donor and acceptor moietieslinked to the probe in the absence of target sequence. Uponhybridization of the probing nucleobase sequence to a target sequence,the efficiency of energy transfer between donor and acceptor moieties ofa PNA Molecular Beacon is altered such that detectable signal from atleast one linked moiety can be used to monitor or quantitate theoccurrence of the hybridization event.

[0033] At a minimum a PNA Molecular Beacon comprises a probingnucleobase sequence, two arm segments, wherein at least one arm segmentis linked to the probe through a flexible linkage, at least one linkeddonor moiety and at least one linked acceptor moiety. The donor andacceptor moieties can be linked at any position within the PNA MolecularBeacon provided that the point of attachment of donor and acceptormoieties of a set are located at opposite ends of the probing nucleobasesequence.

[0034] The probing nucleobase sequence is designed to hybridize to atleast a portion of a target sequence. The first and second arm segmentsof the PNA Molecular Beacon provide for intramolecular or intermolecularinteractions which stabilize secondary structures, dimers and/ormultimers which when formed stabilize the rate of energy transferbetween donor and acceptor moieties of the unhybridized PNA MolecularBeacon. Without intending to be bound to this hypothesis, it is believedthat the flexible linkages provide flexibility and randomness to theotherwise highly structured PNA oligomer thereby resulting in moreefficient energy transfer of the linked donor and acceptor moieties ofthe unhybridized PNA Molecular Beacon as compared with probes of similarnucleobase sequence which do not comprise flexible linkages.

[0035] In one preferred embodiment, this invention is directed to PNAMolecular Beacons comprising an arm segment having a first and secondend. Additionally, there is also a probing nucleobase sequence having afirst and second end wherein, the probing nucleobase sequence iscomplementary or substantially complementary to the target sequence.There is also a second arm segment which is embedded within the probingnucleobase sequence and is complementary or substantially complementaryto the first arm segment. The polymer also comprises a flexible linkagewhich links the second end of the first arm segment to the second end ofthe probing nucleobase sequence. A donor moiety is linked to the firstend of one of either of the first arm segment or the probing nucleobasesequence; and an acceptor moiety is linked to the first end of the otherof either of the first arm segment or the probing nucleobase sequence.

[0036] In still another preferred embodiment, this invention is directedto PNA Molecular Beacons comprising a probing nucleobase sequence havinga first and second end, wherein, the probing nucleobase sequence iscomplementary or substantially complementary to the target sequence.There is also a first arm segment comprising a first and second end anda second arm segment comprising a first and second end, wherein, atleast a portion of the nucleobases of the second arm segment arecomplementary to the nucleobase sequence to the first arm segment. Thepolymer also comprises a first flexible linkage which links the secondend of the first arm segment to either of the first or second end of theprobing nucleobase sequence. There is a second linkage which links thesecond end of the second arm segment to the other of either of the firstor second end of the probing nucleobase sequence. A donor moiety islinked to the first end of one of either of the first or second armsegments; and an acceptor moiety is linked to the first end of the otherof either of the first or the second arm segments.

[0037] In one preferred embodiment, this invention is related to amethod for the detection, identification or quantitation of a targetsequence in a sample. The method comprises contacting the sample with aPNA Molecular Beacon and then detecting, identifying or quantitating thechange in detectable signal associated with at least one donor oracceptor moiety of the probe whereby the change in detectable signal isused to determine the presence, absence or amount of target sequencepresent in the sample of interest. The measurable change in detectablesignal of at least one donor or acceptor moiety of the probe can be usedto determine the presence, absence or amount of target sequence presentin the sample of interest since applicant's have demonstrated that theefficiency of energy transfer between donor and acceptor moieties isaltered by hybridization of the PNA Molecular Beacon to the intendedtarget sequence, under suitable hybridization conditions. Accuratequantitation can be achieved by correcting for signal generated by anyunhybridized PNA Molecular Beacon. Consequently, the PNA MolecularBeacons of this invention are particularly well suited for thedetection, identification or quantitation of target sequences in closedtube assays. Because PNAs are not known to be degraded by enzymes, PNAMolecular Beacons are also particularly well suited for detection,identification or quantitation of target sequences in cells, tissues ororganisms, whether living or not.

[0038] In still another embodiment, this invention is related to kitssuitable for performing an assay which detects the presence, absence ornumber of a target sequences in a sample. The kits of this inventioncomprise one or more PNA Molecular Beacons and other reagents orcompositions which are selected to perform an assay or otherwisesimplify the performance of an assay.

[0039] In yet another embodiment, this invention is also directed to anarray comprising two or more support bound PNA Molecular Beaconssuitable for detecting, identifying or quantitating a target sequence ofinterest. Arrays of PNA Molecular Beacons are convenient because theyprovide a means to rapidly interrogate numerous samples for the presenceof one or more target sequences of interest in real time without using asecondary detection system.

[0040] The methods, kits and compositions of this invention areparticularly useful for the detection of target sequences of organismswhich may be found in food, beverages, water, pharmaceutical products,personal care products, dairy products or environmental samples. Theanalysis of preferred beverages include soda, bottled water, fruitjuice, beer, wine or liquor products. Additionally, the methods,.kitsand compositions will be particularly useful for the analysis of rawmaterials, equipment, products or processes used to manufacture or storefood, beverages, water, pharmaceutical products, personal care productsdairy products or environmental samples.

[0041] Whether support bound or in solution, the methods, kits andcompositions of this invention are particularly useful for the rapid,sensitive, reliable and versatile detection of target sequences whichare particular to organisms which might be found in clinicalenvironments. Consequently, the methods, kits and compositions of thisinvention will be particularly useful for the analysis of clinicalspecimens or equipment, fixtures or products used to treat humans oranimals. For example, the assay may be used to detect a target sequencewhich is specific for a genetically based disease or is specific for apredisposition to a genetically based disease. Non-limiting examples ofdiseases include, β-Thalassemia, sickle cell anemia, Factor-V Leiden,cystic fibrosis and cancer related targets such as p53, p10, BRC-1 andBRC-2.

[0042] In still another embodiment, the target sequence may be relatedto a chromosomal DNA, wherein the detection, identification orquantitation of the target sequence can be used in relation to forensictechniques such as prenatal screening, paternity testing, identityconfirmation or crime investigation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 is a graphical illustration of experimental data.

[0044]FIG. 2 is a graphical illustration of experimental data.

[0045]FIG. 3 is a graphical illustration of experimental data.

[0046]FIG. 4A is an overlay of normalized fluorescence vs. temperatureand absorbance vs. temperature plots for a labeled PNA/PNA bimolecularduplex.

[0047]FIG. 4B is an overlay of normalized fluorescence vs. temperatureand absorbance vs. temperature plots for a labeled unimolecular PNAprobe comprising a flexible linkage.

[0048]FIG. 4C is an overlay of normalized fluorescence vs. temperatureand absorbance vs. temperature plots for a labeled unimolecular PNAprobe which is continuous from the N- to C-terminus.

[0049]FIG. 5 is a graphical representation of comparative fluorescentmelting signal to noise ratios.

[0050]FIG. 6 is an overlay of normalized absorbance vs. temperatureplots for three similar PNA unimolecular probes.

[0051]FIG. 7A is a graphical illustration of data for PNA probes whichexhibit a Type A Fluorescent Thermal Profile.

[0052] FIGS. 7B1, 7B2 and 7B3 are graphical illustrations of data forPNA probes which exhibit a Type B Fluorescent Thermal Profile.

[0053]FIG. 7C is a graphical illustration of data for PNA probes whichexhibit a Type C Fluorescent Thermal Profile.

[0054] FIGS. 8A1, 8A2 and 8A3 are a graphical illustration of data forPNA probes which exhibit a Type A Hybridization Profile.

[0055]FIG. 8B is a graphical illustration of data for PNA probes whichexhibit a Type B Hybridization Profile.

[0056]FIG. 8C is a graphical illustration of data for PNA probes whichexhibit a Type C Hybridization Profile.

[0057]FIG. 9 is an overlay of normalized fluorescence vs. temperatureand absorbance vs. temperature plots for a the labeled unimolecular PNAprobe 0.001.

[0058]FIG. 10 is a graphical illustration of signal to noise dataobtained by Hybridization analysis of PNA oligomers listed in Table 1.

[0059]FIG. 11 is an illustration of several possible hairpinconfigurations of a PNA Molecular Beacon.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] 1. Definitions:

[0061] a. As used herein, the term “nucleobase” shall include thosenaturally occurring and those non-naturally occurring heterocyclicmoieties commonly known to those who utilize nucleic acid technology orutilize peptide nucleic acid technology to thereby generate polymerswhich can sequence specifically bind to nucleic acids.

[0062] b. As used herein, the term “nucleobase sequence” is any segmentof a polymer which comprises nucleobase containing subunits.Non-limiting examples of suitable polymers or polymers segments includeoligonucleotides, oligoribonucleotides, peptide nucleic acids andanalogs or chimeras thereof.

[0063] c. As used herein, the term “target sequence” is any sequence ofnucleobases in a polymer which is sought to be detected. The “targetsequence” may comprise the entire polymer or may be a subsequence of thenucleobase sequence which is unique to the polymer of interest. Withoutlimitation, the polymer comprising the “target sequence” may be anucleic acid, a peptide nucleic acid, a chimera, a linked polymer, aconjugate or any other polymer comprising substituents (e.g.nucleobases) to which the PNA Molecular Beacons of this invention maybind in a sequence specific manner.

[0064] d. As used herein, the term “peptide nucleic acid” or “PNA” shallbe defined as any oligomer, linked polymer or chimeric oligomer,comprising two or more PNA subunits (residues), including any of thecompounds referred to or claimed as peptide nucleic acids in U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or5,786,571 (all of which are herein incorporated by reference). The term“Peptide Nucleic Acid” or “PNA” shall also apply to those nucleic acidmimics described in the following publications: Diderichsen et al.,Tett. Lett. 37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett.7:637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:687-690(1997); Krotz et al., Tett. Lett. 36:6941-6944 (1995); Lagriffoul etal., Bioorg. Med. Chem. Lett. 4:1081-1082 (1994); Lowe et al., J. Chem.Soc. Perkin Trans. 1, (1997) 1:539-546; Lowe et al., J. Chem. Soc.Perkin Trans. 1 1:547-554 (1997); Lowe et al., J. Chem. Soc. PerkinTrans. 1 1:555-560 (1997); and Petersen et al., Bioorg. Med. Chem. Lett.6:793-796 (1996).

[0065] In preferred embodiments, a PNA is a polymer comprising two ormore PNA subunits of the formula:

[0066] wherein, each J is the same or different and is selected from thegroup consisting of H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I. EachK is the same or different and is selected from the group consisting ofO, S, NH and NR¹. Each R¹ is the same or different and is an alkyl grouphaving one to five carbon atoms which may optionally contain aheteroatom or a substituted or unsubstituted aryl group. Each A isselected from the group consisting of a single bond, a group of theformula; —(CJ₂)_(s)— and a group of the formula; —(CJ₂)_(s)C(O)—,wherein, J is defined above and each s is an integer from one to five.The integer t is 1 or 2 and the integer u is 1 or 2. Each L is the sameor different and is independently selected from the group consisting ofJ, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturallyoccurring nucleobase analogs, other non-naturally occurring nucleobases,substituted and unsubstituted aromatic moieties, biotin and fluorescein.In the most preferred embodiment, a PNA subunit consists of a naturallyoccurring or non-naturally occurring nucleobase attached to the azanitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylenecarbonyl linkage.

[0067] 2. Detailed Description

[0068] I. General:

[0069] PNA Synthesis:

[0070] Methods for the chemical assembly of PNAs are well known (See:U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336,5,773,571 or 5,786,571 (all of which are herein incorporated byreference). Chemicals and instrumentation for the support boundautomated chemical assembly of Peptide Nucleic Acids are nowcommercially available. Chemical assembly of a PNA is analogous to solidphase peptide synthesis, wherein at each cycle of assembly the oligomerpossesses a reactive alkyl amino terminus which is condensed with thenext synthon to be added to the growing polymer. Because standardpeptide chemistry is utilized, natural and non-natural amino acids areroutinely incorporated into a PNA oligomer. Because a PNA is apolyamide, it has a C-terminus (carboxyl terminus) and an N-terminus(amino terminus). For the purposes of the design of a hybridizationprobe suitable for antiparallel binding to the target sequence (thepreferred orientation), the N-terminus of the probing nucleobasesequence of the PNA probe is the equivalent of the 5′-hydroxyl terminusof an equivalent DNA or RNA oligonucleotide.

[0071] Labels:

[0072] The labels attached to the PNA Molecular Beacons of thisinvention comprise a set (hereinafter “Beacon Set(s)”) of energytransfer moieties comprising at least one energy donor and at least oneenergy acceptor moiety. Typically, the Beacon Set will include a singledonor moiety and a single acceptor moiety. Nevertheless, a Beacon Setmay contain more than one donor moiety and/or more than one acceptormoiety. The donor and acceptor moieties operate such that one or moreacceptor moieties accepts energy transferred from the one or more donormoieties or otherwise quench signal from the donor moiety or moieties.The energy transfer moieties of this invention operate by both FRET andnon-FRET but preferably do not involve electron transfer.

[0073] Preferably the donor moiety is a fluorophore. Preferredfluorophores are derivatives of fluorescein, derivatives of bodipy,5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), derivativesof rhodamine, Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, texas red and itsderivatives. Though the previously listed fluorophores might alsooperate as acceptors, preferably, the acceptor moiety is a quenchermoiety. Preferably, the quencher moiety is a non-fluorescent aromatic orheteroaromatic moiety. The preferred quencher moiety is4-((-4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl).

[0074] Transfer of energy may occur through collision of the closelyassociated moieties of a Beacon Set or through a nonradiative processsuch as fluorescence resonance energy transfer (FRET). For FRET tooccur, transfer of energy between donor and acceptor moieties of aBeacon Set requires that the moieties be close in space and that theemission spectrum of a donor(s) have substantial overlap with theabsorption spectrum of the acceptor(s) (See: Yaron et al. AnalyticalBiochemistry, 95: 228-235 (1979) and particularly page 232, col. 1through page 234, col. 1). Alternatively, collision mediated(radiationless) energy transfer may occur between very closelyassociated donor and acceptor moieties whether or not the emissionspectrum of a donor moiety(ies) has a substantial overlap with theabsorption spectrum of the acceptor moiety(ies) (See: Yaron et al.,Analytical Biochemistry, 95: 228-235 (1979) and particularly page 229,col. 1 through page 232, col. 1). This process is referred to asintramolecular collision since it is believed that quenching is causedby the direct contact of the donor and acceptor moieties (See: Yaron etal.). As applicant's have demonstrated, the donor and acceptor moietiesattached to the PNA Molecular Beacons of this invention need not have asubstantial overlap between the emission of the donor moieties and theabsorbance of the acceptor moieties. Without intending to be bound tothis hypothesis, this data suggests that collision or contact operatesas the primary mode of quenching in PNA Molecular Beacons.

[0075] Detecting Energy Transfer:

[0076] Because the efficiency of both collision mediated andnonradiative transfer of energy between the donor and acceptor moietiesof a Beacon Set is directly dependent on the proximity of the donor andacceptor moieties, detection of hybrid formation of a PNA MolecularBeacon with a target sequence can be monitored by measuring at least onephysical property of at least one member of the Beacon Set which isdetectably different when the hybridization complex is formed ascompared with when the PNA Molecular Beacon exists in the absence oftarget sequence. We refer to this phenomenon as the self-indicatingproperty of PNA Molecular Beacons. This change in detectable signalresults from the change in efficiency of energy transfer between thedonor and acceptor upon hybridization of the PNA Molecular Beacon to atarget sequence. Preferably, the means of detection will involvemeasuring fluorescence of a donor or acceptor fluorophore of a BeaconSet. Most preferably, the Beacon Set will comprise at least one donorfluorophore and at least one acceptor quencher such that thefluorescence of the donor fluorophore is used to detect, identify orquantitate hybridization.

[0077] PNA Labeling:

[0078] Chemical labeling of a PNA is analogous to peptide labeling.Because the synthetic chemistry of assembly is essentially the same, anymethod commonly used to label a peptide may be used to label a PNA.Typically, the N-terminus of the polymer is labeled by reaction with amoiety having a carboxylic acid group or activated carboxylic acidgroup. One or more spacer moieties can optionally be introduced betweenthe labeling moiety and the probing nucleobase sequence of the oligomer.Generally, the spacer moiety is incorporated prior to performing thelabeling reaction. However, the spacer may be embedded within the labeland thereby be incorporated during the labeling reaction.

[0079] Typically the C-terminal end of the probing nucleobase sequenceis labeled by first condensing a labeled moiety with the support uponwhich the PNA is to be assembled. Next, the first synthon of the probingnucleobase sequence can be condensed with the labeled moiety.Alternatively, one or more spacer moieties can be introduced between thelabeled moiety and the oligomer (e.g. 8-amino-3,6-dioxaoctanoic acid).Once the PNA Molecular Beacon is completely assembled and labeled, it iscleaved from the support deprotected and purified using standardmethodologies.

[0080] The labeled moiety could be a lysine derivative wherein theE-amino group is modified with a donor or acceptor moiety. For examplethe label could be a fluorophore such as 5(6)-carboxyfluorescein or aquencher moiety such as 4-((4-(dimethylamino)phenyl)azo)benzoic acid(dabcyl). Condensation of the lysine derivative with the synthesissupport would be accomplished using standard condensation (peptide)chemistry. The α-amino group of the lysine derivative would then bedeprotected and the probing nucleobase sequence assembly initiated bycondensation of the first PNA synthon with the α-amino group of thelysine amino acid. As discussed above, a spacer moiety could optionallybe inserted between the lysine amino acid and the first PNA synthon bycondensing a suitable spacer (e.g. Fmoc-8-amino-3,6-dioxaoctanoic acid)with the lysine amino acid prior to condensation of the first PNAsynthon of the probing nucleobase sequence.

[0081] Alternatively, a functional group on the assembled, or partiallyassembled, polymer is labeled with a donor or acceptor moiety while itis still support bound. This method requires that an appropriateprotecting group be incorporated into the oligomer to thereby yield areactive functional to which the donor or acceptor moiety is linked buthas the advantage that the label (e.g. dabcyl or a fluorophore) can beattached to any position within the polymer including within the probingnucleobase sequence. For example, the ε-amino group of a lysine could beprotected with a 4-methyl-triphenylmethyl (Mtt), a4-methoxy-triphenylmethyl (MMT) or a 4,4′-dimethoxytriphenylmethyl (DMT)protecting group. The Mtt, MMT or DMT groups can be removed from PNA(assembled using commercially available Fmoc PNA monomers andpolystyrene support having a PAL linker; PerSeptive Biosystems, Inc.,Framingham, Mass.) by treatment of the resin under mildly acidicconditions. Consequently, the donor or acceptor moiety can then becondensed with the ε-amino group of the lysine amino acid. Aftercomplete assembly and labeling, the polymer is then cleaved from thesupport, deprotected and purified using well known methodologies.

[0082] By still another method, the donor or acceptor moiety is attachedto the polymer after it is fully assembled and cleaved from the support.This method is preferable where the label is incompatible with thecleavage, deprotection or purification regimes commonly used tomanufacture the oligomer. By this method, the PNA will generally belabeled in solution by the reaction of a functional group on the polymerand a functional group on the label. Those of ordinary skill in the artwill recognize that the composition of the coupling solution will dependon the nature of oligomer and the donor or acceptor moiety. The solutionmay comprise organic solvent, water or any combination thereof.Generally, the organic solvent will be a polar non-nucleophilic solvent.Non limiting examples of suitable organic solvents include acetonitrile,tetrahydrofuran, dioxane, methyl sulfoxide and N,N′-dimethylformamide.

[0083] Generally the functional group on the polymer to be labeled willbe an amine and the functional group on the label will be a carboxylicacid or activated carboxylic acid. Non-limiting examples of activatedcarboxylic acid functional groups include N-hydroxysuccinimidyl esters.In aqueous solutions, the carboxylic acid group of either of the PNA orlabel (depending on the nature of the components chosen) can beactivated with a water soluble carbodiimide. The reagent,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), is acommercially available reagent sold specifically for aqueous amideforming condensation reactions.

[0084] Generally, the pH of aqueous solutions will be modulated with abuffer during the condensation reaction. Preferably, the pH during thecondensation is in the range of 4-10. When an arylamine is condensedwith the carboxylic acid, preferably the pH is in the range of 4-7. Whenan alkylamine is condensed with a carboxylic acid, preferably the pH isin the range of 7-10. Generally, the basicity of non-aqueous reactionswill be modulated by the addition of non-nucleophilic organic bases.Non-limiting examples of suitable bases include N-methylmorpholine,triethylamine and N,N-diisopropylethylamine. Alternatively, the pH ismodulated using biological buffers such asN-[2-hydroxyethyllpiperazine-N′-[2-ethanesulfonic acid (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium bicarbonate.

[0085] Spacer/Flexible Linker Moieties:

[0086] Spacers are typically used to minimize the adverse effects thatbulky labeling reagents might have on hybridization properties of PNAMolecular Beacons. Flexible linkers typically induce flexibility andrandomness into the PNA Molecular Beacon or otherwise link two or morenucleobase sequences of a probe. Preferred spacer/flexible linkermoieties for probes of this invention consist of one or more aminoalkylcarboxylic acids (e.g. aminocaproic acid) the side chain of an aminoacid (e.g. the side chain of lysine or ornithine) natural amino acids(e.g. glycine), aminooxyalkylacids (e.g. 8-amino-3,6-dioxaoctanoicacid), alkyl diacids (e.g. succinic acid) or alkyloxy diacids (e.g.diglycolic acid). The spacer/linker moieties may also be designed toenhance the solubility of the PNA Molecular Beacon.

[0087] Preferably, a spacer/linker moiety comprises one or more linkedcompounds having the formula: —Y—(O_(m)—(CW₂)_(n))_(o)-Z-. The group Yis a single bond or a group having the formula selected from the groupconsisting of: —(CW₂)_(p)—, —C(O)(CW₂)_(p)—, —C(S)(CW₂)_(p)— and—S(O₂)(CW₂)_(p). The group Z has the formula NH, NR², S or O. Each W isindependently H, R², —OR², F, Cl, Br or I; wherein, each R² isindependently selected from the group consisting of: —CX₃, —CX₂CX₃,—CX₂CX₂CX₃, —CX₂CX(CX₃)₂, and —C(CX₃)₃. Each X is independently H, F,Cl, Br or I. Each m is independently 0 or 1. Each n, o and p areindependently integers from 0 to 10. In the most preferred embodiment,the spacer/flexible linker comprises two linked8-amino-3,6-dioxaoctanoic acid moieties. Consequently,, Y is—C(O)(CW₂)_(p)—, Z is NH, each W is H, m is 1, n is 2, o is 2 and p is1.

[0088] Chimeric Oligomer:

[0089] A chimeric oligomer comprises two or more linked subunits whichare selected from different classes of subunits. For example, a PNA/DNAchimera would comprise at least two PNA subunits linked to at least one2′-deoxyribonucleic acid subunit (For methods and compositions relatedto PNA/DNA chimera preparation See: WO96/40709). The component subunitsof the chimeric oligomers are selected from the group consisting of PNAsubunits, DNA subunits, RNA subunits and analogues thereof.

[0090] Linked Polymer:

[0091] A linked polymer comprises two or more nucleobase sequences whichare linked by a linker. The nucleobase sequences which are linked toform the linked polymer are selected from the group consisting of anoligodeoxynucleotide, an oligoribonucleotide, a peptide nucleic acid anda chimeric oligomer. The PNA probes of this invention include linkedpolymers wherein the probing nucleobase sequence is linked to one ormore additional oligodeoxynucleotide, oligoribonucleotide, peptidenucleic acid or chimeric oligomers.

[0092] Hybridization Conditions/Stringency:

[0093] Those of ordinary skill in the art of nucleic acid hybridizationwill recognize that factors commonly used to impose or controlstringency of hybridization include formamide concentration (or otherchemical denaturant reagent), salt concentration (i.e., ionic strength),hybridization temperature, detergent concentration, pH and the presenceor absence of chaotropes. Optimal stringency for a probing nucleobasesequence/target sequence combination is often found by the well knowntechnique of fixing several of the aforementioned stringency factors andthen determining the effect of varying a single stringency factor. Thesame stringency factors can be modulated to thereby control thestringency of hybridization of PNA Molecular Beacons to targetsequences, except that the hybridization of a PNA is fairly independentof ionic strength. Optimal stringency for an assay may be experimentallydetermined by examination of each stringency factor until the desireddegree of discrimination is achieved.

[0094] Probing Nucleobase Sequence:

[0095] The probing nucleobase sequence of a PNA Molecular Beacon is thesequence recognition portion of the construct. Therefore, the probingnucleobase sequence is designed to hybridize to at least a portion ofthe target sequence. Preferably the probing nucleobase sequencehybridizes to the entire target sequence. The probing nucleobasesequence is a non-polynucleotide and preferably the probing nucleobasesequence is composed exclusively of PNA subunits. The subunit length ofthe probing nucleobase sequence will therefore generally be chosen suchthat a stable complex is formed between the PNA Molecular Beacon and thetarget sequence sought to be detected, under suitable hybridizationconditions. The probing nucleobase sequence of a PNA oligomer, suitablefor the practice of this invention, will generally have a length ofbetween 5 and 30 PNA subunits. Preferably, the probing nucleobasesequence will be 8 to 18 subunits in length. Most preferably, theprobing nucleobase sequence will be 11-17 subunits in length.

[0096] The probing nucleobase sequence of a PNA Molecular Beacons willgenerally have a nucleobase sequence which is complementary to thetarget sequence. Alternatively, a substantially complementary probingsequence might be used since it has been demonstrated that greatersequence discrimination can be obtained when utilizing probes whereinthere exists a single point mutation (base mismatch) between the probingnucleobase sequence and the target sequence (See: Guo et al., NatureBiotechnology 15: 331-335 (1997), Guo et al., WO97/46711; and Guo etal., U.S. Pat. No. 5,780,233, herein incorporated by reference).

[0097] Arm Segments

[0098] The arm segments of the PNA Molecular Beacon are designed toanneal to each other and thereby stabilize the interactions which fixthe energy transfer of linked donor and acceptor moieties until the PNAMolecular Beacon hybridizes to the target sequence. The arm segments maybe of different lengths, but, are preferably the same length. Thepreferred length of the arm segments will depend on the stabilitydesired for the interactions. However, the arm segments must not be solong that they prohibit hybridization to the target sequence.Preferably, the arm segments are 2-6 subunits in length and mostpreferably the arm segments are 2-5 subunits in length since applicant'sdata demonstrates that the highest signal to noise ratios are obtainedwith PNA Molecular Beacons having arm segments of 5 or less subunits.Preferably arm segments of a PNA Molecular Beacon are comprisedprimarily of PNA subunits and preferably comprised of only PNA subunits.However, salt pairs and hydrophobic/hydrophobic interactions maycontribute to the stability of the interactions which fix the proximityof the donor and acceptor moieties

[0099] In certain embodiments, both arm segments are external to theprobing nucleobase sequence (See: FIG. 11; Configuration III).Alternatively, one arm segment may be embedded within a probingnucleobase sequence (See: FIG. 11; Configurations I and II). When onearm segment is embedded within the probing nucleobase sequence,preferably the other arm segment is oriented to the N-terminus of thePNA Molecular Beacon and the probing nucleobase sequence is orientedtoward the C-terminus of the PNA Molecular Beacon.

[0100] Flexible Linkages

[0101] The flexible linkages link one or more arm forming segments tothe PNA Molecular Beacon. Without intending to be bound to thishypothesis, it is believed that flexible linkages provide flexibilityand randomness to the otherwise highly structured PNA oligomer therebyresulting in more efficient energy transfer of the linked donor andacceptor moieties of the unhybridized PNA Molecular Beacon. The lengthand composition of the flexible linkages will be judiciously chosen tofacilitate intramolecular interactions between functional groups of thepolymer (e.g. nucleobase-nucleobase interactions) which would otherwisenot be able to freely interact. Flexible linkages appear to produce PNAMolecular Beacons which exhibit higher signal to noise ratios inhybridization assays and a more reversible modulation of fluorescentsignal in response to thermal changes in environment as compared withPNA Molecular Beacons which do not possess flexible linkages. Thus,flexible linkages are an important feature of the PNA Molecular Beaconsof this invention.

[0102] Blocking Probes:

[0103] Blocking probes are PNA or nucleic acid probes which can be usedto suppress the binding of the probing nucleobase sequence of a probe toa hybridization site which is unrelated or closely related to the targetsequence (See: Coull et al., PCT/US97/21845, a.k.a. WO98/24933).Generally, the blocking probes suppress the binding of the probingnucleobase sequence to closely related non-target sequences because theblocking probe hybridizes to the non-target sequence to form a morethermodynamically stable complex than is formed by hybridization betweenthe probing nucleobase sequence and the non-target sequence. Thus,blocking probes are typically unlabeled probes used in an assay tothereby suppress non-specific signal. Because they are usually designedto hybridize to closely related non-target sequence sequences, typicallya set of two or more blocking probes will be used in an assay to therebysuppress non-specific signal from non-target sequences which could bepresent and interfere with the performance of the assay.

[0104] II. Preferred Embodiments of the Invention:

[0105] PNA Molecular Beacons:

[0106] Tyagi et al. and Tyagi2 et al. disclose nucleic acid MolecularBeacons which comprise a hairpin loop and stem to which energy transferdonor and acceptor moieties are linked at opposite ends of the nucleicacid polymer. Numerous PNA polymers were examined in an attempt toprepare a PNA Molecular Beacon. The applicant's have determined that allprobes they examined, which contained linked donor and acceptor moietiesexhibited a low inherent noise (background) and an increase indetectable signal upon binding of the probe to a target sequence. Verysurprisingly, these characteristic properties of a nucleic acidMolecular Beacon were observed whether or not the PNA oligomer possessedself-complementary arm segments intended to form a PNA hairpin. Forexample, PNA oligomers prepared as control samples which by design didnot possess any self-complementary arm segments suitable for forming ahairpin exhibited a signal (PNA oligomer bound to target sequence) tonoise (no target sequence present) ratio which was quite favorable ascompared with probes comprising flexible linkages and self-complementaryarm segments.

[0107] Applicant's data further demonstrates that flexible linkagesinserted within the probe and shorter self-complementary arm segmentsare a preferred embodiment since the signal to noise ratio of probes ofthis embodiment compare well with the signal to noise ratio publishedfor nucleic acid hairpins (approximately 25 to 1). The data compiled byapplicants is inconclusive with respect to whether or not the PNAMolecular Beacons they prepared which have shorter arms segments (2-5subunits in length) and one or more flexible linkages exist as hairpins.However, applicant's data demonstrates that probes with longer armsegments (e.g. 9 subunits) do form a hairpin (See: Example 19 of thisspecification) and unlabeled probes having arms segments as short as sixsubunits do not exist primarily as a hairpin (See: Example 19 of thisspecification). Furthermore, the signal to noise ratio for those theprobes having longer arm segments suitable for forming a hairpinexhibited very poor a signal to noise ratios upon melting of the hairpinor when in the presence of a complementary nucleic acid. Consequently,embodiments having longer arm segments (e.g. 6 or more subunits) do notappear to be well suited for use in the detection of nucleic acidtargets.

[0108] This invention pertains to methods, kits and compositionspertaining to PNA Molecular Beacons. Though we refer to the probes ofthis invention as PNA Molecular Beacons, we do not mean to imply thatthey exist as hairpins since they may well exist as aggregates,bimolecular constructs or as higher order hybrids (e.g. multimers).Regardless of the nature of the secondary structure, a PNA MolecularBeacon efficiently transfers energy between donor and acceptor moietieslinked to the probe in the absence of target sequence. Uponhybridization of the probing nucleobase sequence to a target sequence,the efficiency of energy transfer between donor and acceptor moieties ofa PNA Molecular Beacon is altered such that detectable signal from atleast one linked moiety can be used to monitor or quantitate theoccurrence of the hybridization event.

[0109] Generally, a PNA Molecular Beacon is a polymer suitable fordetecting, identifying or quantitating a target sequence. At a minimum,a PNA Molecular Beacon comprises a probing nucleobase sequence, two armsegments, wherein at least one arm segment is linked to the probethrough a flexible linkage, at least one linked donor moiety and atleast one linked acceptor moiety. The donor and acceptor moieties can belinked at any position within the PNA Molecular Beacon provided they areseparated by at least a portion of the probing nucleobase sequence.Preferably the donor and acceptor moieties of a Beacon Set are locatedat opposite ends of the probing nucleobase sequence and most preferablyat the termini of the PNA Molecular Beacon. The PNA Molecular Beacon isfurther characterized in that the probe exhibits detectable change in atleast one property of at least one linked donor or acceptor moiety whichoccurs upon hybridization to the target sequence under suitablehybridization conditions.

[0110] In one preferred embodiment, this invention is directed to a PNAMolecular Beacons comprising an arm segment having a first and secondend. Additionally, there is also a probing nucleobase sequence having afirst and second end wherein, the probing nucleobase sequence iscomplementary or substantially complementary to the target sequence.There is also a second arm segment which is embedded within the probingnucleobase sequence and is complementary or substantially complementaryto the first arm segment. The polymer also comprises a flexible linkagewhich links the second end of the first arm segment to the second end ofthe probing nucleobase sequence. A donor moiety is linked to the firstend of one of either of the first arm segment or the probing nucleobasesequence; and an acceptor moiety is linked to the first end of the otherof either of the first arm segment or the probing nucleobase sequence.

[0111] In still another preferred embodiment, this invention is directedto a PNA Molecular Beacon comprising a probing nucleobase sequencehaving a first and second end, wherein, the probing nucleobase sequenceis complementary or substantially complementary to the target sequence.There is also a first arm segment comprising a first and second end anda second arm segment comprising a first and second end, wherein, atleast a portion of the nucleobases of the second arm segment arecomplementary to the nucleobase sequence to the first arm segment. Thepolymer also comprises a first flexible linkage which links the secondend of the first arm segment to either of the first or second end of theprobing nucleobase sequence. There is a second linkage which links thesecond end of the second arm segment to the other of either of the firstor second end of the probing nucleobase sequence. A donor moiety islinked to the first end of one of either of the first or second armsegments; and an acceptor moiety is linked to the first end of the otherof either of the first or the second arm segments.

[0112] Preferably, a PNA Molecular Beacons is assembled by stepwisecondensation of suitably protected amino acid moieties. Consequently,the polymer is preferably continuous from the amino to the carboxylterminus. In the most preferred configuration, PNA Molecular Beacons arecontinuous from the N-terminus to the C-terminus wherein the first armsegment is oriented toward the N-terminus and the probing nucleobasesequence is oriented toward the C-terminus of the polymer. Additionally,the preferred PNA Molecular Beacons comprise a probing nucleobasesequence which is perfectly complementary to the target sequence and afirst arm segment which is perfectly complementary to the second armsegment.

[0113] It is not a requirement that the PNA Molecular Beacons of thisinvention form a hairpin. However, if hairpins are formed, preferredembodiments of the PNA Molecular Beacons of this invention can generallybe represented in three configurations with are illustrated in FIG. 11.In configuration I, the probing nucleobase sequence is located at thecarboxyl terminus of the polymer. The probing nucleobase sequence islinked to the arm forming segment through one or more flexible linkermoieties. In this embodiment, one of the two arm segments is embeddedwithin the probing nucleobase sequence. As illustrated, the donor andacceptor moieties are located at opposite ends of the PNA MolecularBeacon but either orientation of the labels is acceptable. Thisembodiment of a Molecular Beacon is unique even in light of the nucleicacid Molecular Beacons, because one of the two arm forming segments isembedded within the probing segment. Minimization of sequence length ispreferred since it should reduce non-specific interactions.

[0114] In configuration II, the positioning of the probing nucleobasesequence and arm segments are inverted as compared with configuration I.In this configuration the probing nucleobase sequence is located at theamino terminus of the polymer and is linked to an arm forming segmentthrough one or more flexible linker moieties. As illustrated, the donorand acceptor moieties are located at opposite termini of the PNAMolecular Beacon but either orientation of the labels is acceptable.

[0115] In configuration III, the entire probing nucleobase sequence isexternal to the two arm forming segments. Thus, this embodiment is moresimilar to the nucleic acid Molecular Beacons than is eitherconfiguration I or II. Configuration III, however, differs from nucleicacid Molecular Beacons in that it is comprised of PNA subunits and alsocontains at least one flexible linkage separating a probing nucleobasesequence and the arm segments.

[0116] Unique Features of PNA Molecular Beacons:

[0117] There are many differences between prior art nucleic acidconstructs and the PNA Molecular Beacons of this invention. For example,nucleic acid constructs comprise a polynucleotide backbone whereas thePNA Molecular Beacons of this invention comprise a probing nucleobasesequence which is not a polynucleotide. Thus, PNA Molecular Beaconswhich comprise PNA subunits exhibit all of the favorable properties ofPNA such as resistance to nuclease degradation, salt independentsequence hybridization to complementary nucleic acids and rapidhybridization kinetics. For probes which do form hairpin stems, the Tmof the stem duplex is substantially independent of the presence orabsence of magnesium and the ionic strength of the environment.

[0118] Additionally, several of the constructs designed by applicantsare PNA Molecular Beacons having arm segments which are embedded withinthe probing nucleobase sequence. These unique constructs are shorterthan corresponding nucleic acid Molecular Beacons. Shorter probes areless costly to synthesize, are generally easier to purify and shouldexhibit few non-specific interactions since they will comprise lessnucleobase sequence diversity.

[0119] Additionally, the constructs described herein comprise flexiblelinkages which applicants have demonstrated to be a preferred embodimentsince a higher signal to noise ratio is achieved as compared with PNAprobes of similar subunit design which do not comprise flexiblelinkages. Similarly, the preferred PNA Molecular Beacons of thisinvention comprise short arm segments since applicant's datademonstrates a clear inverse correlation between arm length and signalto noise ratio. The preferred PNA Molecular Beacons of this inventioncomprise arms sequences of five or less, and more preferably three orless, subunits.

[0120] Probe Sets:

[0121] In another embodiment, this invention is directed to sets of PNAMolecular Beacons suitable for detecting or identifying the presence,absence or amount of two or more different target sequences which mightbe present in a sample. The characteristics of PNA Molecular Beaconssuitable for the detection, identification or quantitation of targetsequences have been previously described herein. The grouping of PNAMolecular Beacons within sets characterized for specific detection oftwo or more target sequences is a preferred embodiment of thisinvention.

[0122] Probe sets of this invention shall comprise at least one PNAMolecular Beacon but need not comprise only PNA Molecular Beacons. Forexample, probe sets of this invention may comprise mixtures of PNAMolecular Beacons, other PNA probes and/or nucleic acid probes, providedhowever that a set comprises at least one PNA Molecular Beacon asdescribed herein. In preferred embodiments, at least one probe of theset is a blocking probe, as defined herein.

[0123] Immobilization of a PNA Molecular Beacon to a Surface:

[0124] One or more PNA Molecular Beacons may optionally be immobilizedto a surface. In one embodiment, the probe can be immobilized to thesurface using the well known process of UV-crosslinking. Alternatively,the PNA oligomer is synthesized on the surface in a manner suitable fordeprotection but not cleavage from the synthesis support.

[0125] Preferably, the probe is covalently linked to a surface by thereaction of a suitable functional groups on the probe and support.Functional groups such as amino groups, carboxylic acids and thiols canbe incorporated in a PNA Molecular Beacon by extension of one of thetermini with suitable protected moieties (e.g. lysine, glutamic acid andcystine). When extending the terminus, one functional group of abranched amino acid such as lysine can be used to incorporate the donoror acceptor label at the appropriate position in the polymer (See:Section entitled “PNA Labeling”) while the other functional group of thebranch is used to optionally further extend the polymer and immobilizeit to a surface.

[0126] Methods for the attachment of probes to surfaces generallyinvolve the reaction of a nucleophilic group, (e.g. an amine or thiol)of the probe to be immobilized, with an electrophilic group on thesupport to be modified. Alternatively, the nucleophile can be present onthe support and the electrophile (e.g. activated carboxylic acid)present on the PNA Molecular Beacon. Because native PNA possesses anamino terminus, a PNA will not necessarily require modification tothereby immobilize it to a surface (See: Lester et al., Poster entitled“PNA Array Technology”).

[0127] Conditions suitable for the immobilization of a PNA to a surfacewill generally be similar to those conditions suitable for the labelingof a PNA (See: subheading “PNA Labeling”). The immobilization reactionis essentially the equivalent of labeling the PNA whereby the label issubstituted with the surface to which the PNA probe is to be covalentlyimmobilized.

[0128] Numerous types of surfaces derivatized with amino groups,carboxylic acid groups, isocyantes, isothiocyanates and malimide groupsare commercially available. Non-limiting examples of suitable surfacesinclude membranes, glass, controlled pore glass, polystyrene particles(beads), silica and gold nanoparticles.

[0129] When immobilized to a surface, energy transfer between moietiesof a Beacon Set will occur in the PNA Molecular Beacon. Uponhybridization to a target sequence under suitable hybridizationconditions, the location on the surface where the PNA Molecular Beacon(of known sequence) is attached will generate detectable signal based onthe measurable change in signal of at least one member of the Beacon Setof the immobilized PNA Molecular Beacon. Consequently, the intensity ofthe signal on the surface can be used to detect, identify or quantitatethe presence or amount of a target sequence in a sample which contactsthe surface to which the PNA Molecular Beacon is immobilized. In apreferred embodiment, detection of surface fluorescence will be used todetect hybridization to a target sequence.

[0130] Detectable and Independently Detectable Moieties/MultiplexAnalysis:

[0131] In preferred embodiments of this invention, a multiplexhybridization assay is performed. In a multiplex assay, numerousconditions of interest are simultaneously examined. Multiplex analysisrelies on the ability to sort sample components or the data associatedtherewith, during or after the assay is completed. In preferredembodiments of the invention, distinct independently detectable moietiesare used to label the different PNA Molecular Beacons of a set. Theability to differentiate between and/or quantitate each of theindependently detectable moieties provides the means to multiplex ahybridization assay because the data which correlates with thehybridization of each of the distinctly (independently) labeled PNAMolecular Beacons to a target sequence can be correlated with thepresence, absence or quantity of the target sequence sought to bedetected in a sample. Consequently, the multiplex assays of thisinvention may be used to simultaneously detect the presence, absence oramount of one or more target sequences which may be present in the samesample in the same assay. Preferably, independently detectablefluorophores will be used as the independently detectable moieties of amultiplex assay using PNA Molecular Beacons. For example, two PNAMolecular Beacons might be used to detect each of two different targetsequences wherein a fluorescein (green) labeled probe would be used todetect the first of the two target sequences and a rhodamine or Cy3(red) labeled probe would be used to detect the second of the two targetsequences. Consequently, a green, a red or a green and red signal in theassay would signify the presence of the first, second and first andsecond target sequences, respectively.

[0132] Arrays of PNA Molecular Beacons:

[0133] Arrays are surfaces to which two or more probes of interest havebeen immobilized at predetermined locations. Arrays comprising bothnucleic acid and PNA probes have been described in the literature. Theprobe sequences immobilized to the array are judiciously chosen tointerrogate a sample which may contain one or more target sequences ofinterest. Because the location and sequence of each probe is known,arrays are generally used to simultaneously detect, identify orquantitate the presence or amount of one or more target sequences in thesample. Thus, PNA arrays may be useful in diagnostic applications or inscreening compounds for leads which might exhibit therapeutic utility.

[0134] For example, in a diagnostic assay a target sequence is capturedby the complementary probe on the array surface and then theprobe/target sequence complex is detected using a secondary detectionsystem. In one embodiment the probe/target sequence complex is detectedusing a second probe which hybridizes to another sequence of the targetmolecule of interest. In another embodiment, a labeled antibody is usedto detect, identify or quantitate the presence of the probe/targetsequence complex.

[0135] Since the composition of the PNA Molecular Beacon is known at thelocation on the surface of the array (because the PNA was synthesized orattached to this position in the array), the composition of targetsequence(s) can be directly detected, identified or quantitated bydetermining the location of detectable signal generated in the array.Because hybridization of the PNA Molecular Beacon to a target sequenceis self-indicating, no secondary detection system is needed to analyzethe array for hybridization between the PNA Molecular Beacon and thetarget sequence.

[0136] Arrays comprised of PNAs have the additional advantage that PNAsare highly stable and should not be degraded by enzymes which degradenucleic acid. Therefore, PNA arrays should be reusable provided thenucleic acid from one sample can be striped from the array prior tointroduction of the second sample. Upon stripping of hybridized targetsequences, signal on the array of PNA Molecular Beacons should againbecome reduced to background. Because PNAs are not degraded by heat orendonuclease and exonuclease activity, arrays of PNA Molecular Beaconshould be suitable for simple and rapid regeneration by treatment withheat, nucleases or chemical denaturants such as aqueous solutionscontaining formamide, urea and/or sodium hydroxide.

[0137] Methods:

[0138] In yet another embodiment, this invention is directed to a methodfor the detection, identification or quantitation of a target sequencein a sample. The method comprises contacting the sample with a PNAMolecular Beacon and then detecting, identifying or quantitating thechange in detectable signal associated with at least one moiety of aBeacon Set whereby correlation between detectable signal andhybridization is possible since PNA Molecular Beacons areself-indicating. Because PNA Molecular Beacons are self-indicating, thismethod is particularly well suited to analysis performed in a closedtube assay (a.k.a. “homogeneous assays”). By closed tube assay we meanthat once the components of the assay have been combined, there is noneed to open the tube or remove contents of the assay to determine theresult. Since the tube need not, and preferably will not, be opened todetermine the result, there must be some detectable or measurable changewhich occurs and which can be observed or quantitated without openingthe tube or removing the contents of the assay. Thus, most closed tubeassays rely on a change in fluorescence which can be observed with theeye or otherwise be detected and/or quantitated with a fluorescenceinstrument which uses the tube as the sample holder. Examples of suchinstruments include the Light Cycler from Idaho Technologies and thePrism 7700 from Perkin Elmer.

[0139] Preferred closed tube assays of this invention comprise thedetection of nucleic acid target sequences which have been synthesizedor amplified by operation of the assay. Non-limiting examples ofpreferred nucleic acid synthesis or nucleic acid amplification reactionsare Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), StrandDisplacement Amplification (SDA), Transcription-Mediated Amplification(TMA), Rolling Circle Amplification (RCA) and Q-beta replicase. The PNAMolecular Beacons present in the closed tube assay will generatedetectable signal in response to target sequence production from thenucleic acid synthesis or nucleic acid amplification reaction occurringin the closed tube assay. In a most preferred embodiment, the assay isan asymmetric PCR reaction.

[0140] Because the PNA Molecular Beacons of this invention can bedesigned to be stable to the enzymes found in the cell, this method isparticularly well suited to detecting a target sequence in a cell,tissue or organism, whether living or not. Thus, in preferredembodiments, in-situ hybridization is used as the assay format fordetecting identifying or quantitating target organisms. Most preferably,fluorescence in-situ hybridization (FISH or PNA-FISH) is the assayformat. Exemplary methods for performing PNA-FISH can be found in:Thisted et al. Cell Vision, 3:358-363 (1996) or WIPO Patent ApplicationWO97/18325, herein incorporated by reference.

[0141] Organisms which have been treated with the PNA Molecular Beaconsof this invention can be detected by several exemplary methods. Thecells can be fixed on slides and then visualized with a microscope orlaser scanning device. Alternatively, the cells can be fixed and thenanalyzed in a flow cytometer (See for example: Lansdorp et al.; WIPOPatent Application; WO97/14026). Slide scanners and flow cytometers areparticularly useful for rapidly quantitating the number of targetorganisms present in a sample of interest.

[0142] Because the method of this invention may be used in a probe-basedhybridization assay, this invention will find utility in improvingassays used to detect, identify of quantitate the presence or amount ofan organism or virus in a sample through the detection of targetsequences associated with the organism or virus. (See: U.S. Pat. No.5,641,631, entitled “Method for detecting, identifying and quantitatingorganisms and viruses” herein incorporated by reference). Similarly,this invention will also find utility in an assay used in the detection,identification or quantitation of one or more species of an organism ina sample (See U.S. Pat. No. 5,288,611, entitled “Method for detecting,identifying and quantitating organisms and viruses” herein incorporatedby reference). This invention will also find utility in an assay used todetermine the effect of antimicrobial agents on the growth of one ormore microorganisms in a sample (See: U.S. Pat. No. 5,612,183, entitled“Method for determining the effect of antimicrobial agents on growthusing ribosomal nucleic acid subunit subsequence specific probes” hereinincorporated by reference). This invention will also find utility in anassay used to determine the presence or amount of a taxonomic group oforganisms in a sample (See: U.S. Pat. No. 5,601,984, entitled “Methodfor detecting the presence of amount of a taxonomic group of organismsusing specific r-RNA subsequences as probes” herein incorporated byreference).

[0143] When performing the method of this invention, it may bepreferable to use one or more unlabeled or independently detectableprobes in the assay to thereby suppress the binding of the PNA MolecularBeacon to a non-target sequence. The presence of the “blocking probe(s)”helps to increase the discrimination of the assay and thereby improvereliability and sensitivity (signal to noise ratio).

[0144] In certain embodiments of this invention, one target sequence isimmobilized to a surface by proper treatment of the sample.Immobilization of the nucleic acid can be easily accomplished byapplying the sample to a membrane and then UV-crosslinking. For example,the samples may be arranged in an array so that the array can besequentially interrogated with one or more PNA Molecular Beacons tothereby determine whether each sample contains one or more targetsequence of interest.

[0145] In still another embodiment, the PNA Molecular Beacon isimmobilized to a support and the samples are sequentially interrogatedto thereby determine whether each sample contains a target sequence ofinterest. In preferred embodiments, the PNA Molecular Beacons areimmobilized on an array which is contacted with the sample of interest.Consequently, the sample can be simultaneously analyzed for the presenceand quantity of numerous target sequences of interest wherein thecomposition of the PNA Molecular Beacons are judiciously chosen andarranged at predetermined locations on the surface so that the presence,absence or amount of particular target sequences can be unambiguouslydetermined. Arrays of PNA Molecular Beacons are particularly usefulbecause no second detection system is required since PNA MolecularBeacons are self-indicating. Consequently, this invention is alsodirected to an array comprising two or more support bound PNA MolecularBeacons suitable for detecting, identifying or quantitating a targetsequence of interest.

[0146] Kits:

[0147] In yet another embodiment, this invention is directed to kitssuitable for performing an assay which detects the presence, absence oramount of one or more target sequence which may be present in a sample.The characteristics of PNA Molecular Beacons suitable for the detection,identification or quantitation of amount of one or more target sequencehave been previously described herein. Furthermore, methods suitable forusing the PNA Molecular Beacon components of a kit to detect, identifyor quantitate one or more target sequence which may be present in asample have also been previously described herein.

[0148] The kits of this invention comprise one or more PNA MolecularBeacons and other reagents or compositions which are selected to performan assay or otherwise simplify the performance of an assay. Preferredkits contain sets of PNA Molecular Beacons, wherein each of at least twoPNA Molecular Beacons of the set are used to distinctly detect anddistinguish between the two or more different target sequences which maybe present in the sample. Thus, the PNA Molecular Beacons of the set arepreferably labeled with independently detectable moieties so that eachof the two or more different target sequences can be individuallydetected, identified or quantitated (a multiplex assay).

[0149] Exemplary Applications for Using the Invention:

[0150] Whether support bound or in solution, the methods, kits andcompositions of this invention are particularly useful for the rapid,sensitive, reliable and versatile detection of target sequences whichare particular to organisms which might be found in food, beverages,water, pharmaceutical products, personal care products, dairy productsor environmental samples. The analysis of preferred beverages includesoda, bottled water, fruit juice, beer, wine or liquor products.Consequently, the methods, kits and compositions of this invention willbe particularly useful for the analysis of raw materials, equipment,products or processes used to manufacture or store food, beverages,water, pharmaceutical products, personal care products, dairy productsor environmental samples.

[0151] Whether support bound or in solution, the methods, kits andcompositions of this invention are particularly useful for the rapid,sensitive, reliable and versatile detection of target sequences whichare particular to organisms which might be found in clinicalenvironments. Consequently, the methods, kits and compositions of thisinvention will be particularly useful for the analysis of clinicalspecimens or equipment, fixtures or products used to treat humans oranimals. For example, the assay may be used to detect a target sequencewhich is specific for a genetically based disease or is specific for apredisposition to a genetically based disease. Non-limiting examples ofdiseases include, β-Thalassemia, sickle cell anemia, Factor-V Leiden,cystic fibrosis and cancer related targets such as p53, p10, BRC-1 andBRC-2.

[0152] In still another embodiment, the target sequence may be relatedto a chromosomal DNA, wherein the detection, identification orquantitation of the target sequence can be used in relation to forensictechniques such as prenatal screening, paternity testing, identityconfirmation or crime investigation.

EXAMPLES

[0153] This invention is now illustrated by the following examples whichare not intended to be limiting in any way.

Example 1

[0154] Synthesis of N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OH

[0155] To 20 mmol of N-α-(Fmoc)-N-ε-(t-boc)-L-lysine-OH was added 60 mLof 2/1 dichloromethane (DCM)/trifluoroacetic acid (TFA). The solutionwas allowed to stir until the tert-butyloxycarbonyl (t-boc) group hadcompletely been removed from the N-α-(Fmoc)-N-ε-(t-boc)-L-lysine-OH. Thesolution was then evaporated to dryness and the residue redissolved in15 mL of DCM. An attempt was then made to precipitate the product bydropwise addition of the solution to 350 mL of ethyl ether. Because theproduct oiled out, the ethyl ether was decanted and the oil put underhigh vacuum to yield a white foam. The white foam was dissolved in 250mL of water and the solution was neutralized to pH 4 by addition ofsaturated sodium phosphate (dibasic). A white solid formed and wascollected by vacuum filtration. The product was dried in a vacuum ovenat 35-40° C. overnight. Yield 17.6 mmol, 88%.

Example 2

[0156] Synthesis of N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH

[0157] To 1 mmol of N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OH (Example 1) wasadded 5 mL of N,N′-dimethylformamide (DMF) and 1.1 mmol of TFA. Thissolution was allowed to stir until the amino acid had completelydissolved.

[0158] To 1.1 mmol of 4-((4-(dimethylamino)phenyl)azo)benzoic acid,succinimidyl ester (Dabcyl-NHS; Molecular Probes, P/N D-2245) was added4 mL of DMF and 5 mmol of diisopropylethylamine (DIEA). To this stirringsolution was added, dropwise, the N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OHsolution prepared as described above. The reaction was allowed to stirovernight and was then worked up.

[0159] The solvent was vacuum evaporated and the residue partitioned in50 mL of DCM and 50 mL of 10% aqueous citric acid. The layers wereseparated and the organic layer washed with aqueous sodium bicarbonateand again with 10% aqueous citric acid. The organic layer was then driedwith sodium sulfate, filtered and evaporated to an orange foam. The foamwas crystallized from acetonitrile (ACN) and the crystals collected byvacuum filtration. Yield 0.52 mmol, 52%.

Example 3

[0160] Synthesis of N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-PAL-Peg/PSSynthesis Support

[0161] The N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH (Example 2) was used toprepare a synthesis support useful for the preparation of C-terminaldabcylated PNAs. The fluorenylmethoxycarbonyl (Fmoc) group of 0.824 g ofcommercially available Fmoc-PAL-Peg-PS synthesis support (PerSeptiveBiosystems, Inc.; P/N GEN913384) was removed by treatment, in a flowthrough vessel, with 20% piperidine in DCM for 30 minutes. The supportwas then washed with DCM. Finally, the support was washed with DMF anddried with a flushing stream of argon.

[0162] A solution containing 0.302 gN-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH, 3.25 mL of DMF, 0.173 g[O-(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), 0.101 mL DIEA and 0.068 mL 2,6-lutidine wasprepared by sequential combination of the reagents. This solution wasthen added to the washed synthesis support and allowed to react for 2hours. The solution was then flushed through the vessel with a stream ofargon and the support washed sequentially with DMF, DCM and DMF. Theresin was then dried with a stream of argon.

[0163] The support was then treated with 5 mL of standard commerciallyavailable PNA capping reagent (PerSeptive Biosystems, Inc., P/NGEN063102). The capping reagent was then flushed from the vessel and thesupport was washed with DMF and DCM. The support was then dried with astream of argon. Finally, the synthesis support was dried under highvacuum.

[0164] Final loading of the support was determined by analysis of Fmocloading of three samples of approximately 6-8 mg. Analysis determinedthe loading to be approximately 0.145 mmol/g.

[0165] This synthesis support was packed into an empty PNA synthesiscolumn, as needed, and used to prepare PNA oligomers having a C-terminaldabcyl quenching moiety attached to the PNA oligomer through the ε-aminogroup of the C-terminal L-lysine amino acid.

Example 4

[0166] Synthesis Of PNA

[0167] PNAs were synthesized using commercially available reagents andinstrumentation obtained from PerSeptive Biosystems, Inc. Doublecouplings were routinely performed to improve the quality of the crudeproduct. PNAs possessing a C-terminal dabcyl moiety were prepared byperforming the synthesis using the dabcyl-lysine modified synthesissupport prepared as described in Example 3 or by labeling the N-ε-aminogroup of the C-terminal lysine residue while the PNA was still supportbound as described in Example 10. All PNAs possessing both an N-terminalfluorescein moiety, as well as, a C-terminal dabcyl moiety were treatedwith the appropriate labeling reagents and linkers (as required) priorto cleavage from the synthesis support.

Example 5

[0168] Preferred Method for Removal of the Fmoc Protecting Group

[0169] The synthesis support was treated with a solution of 25%piperidine in DMF for 5-15 minutes at room temperature. After treatment,the synthesis support was washed and dried under high vacuum. Thesupport was then treated with the appropriate labeling reagent and/orcleaved from the synthesis support.

Example 6

[0170] Synthesis of Fluorescein-O-Linker

[0171] To 7.5 mmol ofN-(tert-butyloxycarbonyl)-8-amino-3,6-dioxaoctanoic acid stirring in 10mL of DCM was added 50 mmol of TFA. The solution was stirred at roomtemperature until the t-boc group was completely removed. The solventwas then removed by vacuum evaporation and the product was thenresuspended in 10 mL of DCM.

[0172] To this stirring solution was added, dropwise, a solutioncontaining 7.5 mmol of Di-O-pivaloyl-5(6)-carboxyfluorescein-NHS ester,30 mmol of N-methylmorpholine (NMM) and 20 mL of DCM. The reaction wasallowed to run overnight and was then transferred to a separatory funnelin the morning.

[0173] This organic solution was washed with aqueous 10% citric acid twotimes and then dried with sodium sulfate, filtered and evaporated to abrown foam. The product was column purified using silica gel. A DCMmobile phase and stepwise methanol gradient was used to elute theproduct from the stationary phase. Yield 2.8 g of foam which wasprecipitated by dissolution in a minimal amount of DCM and dropwiseaddition of that solution to hexane. Yield 2.32 g white powder. Thepurity of the product was not suitable for labeling so an additionalreversed phase chromatographic separation was performed on a sample ofthis material.

[0174] One gram of the precipitated product was dissolved in 30 mL of a50 mM aqueous-triethylammonium acetate (pH 7) containing 40%acetonitrile. This solution was then added to a pre-equilibrated 2 gWaters Sep-Pack Vac 12 cc tC18 cartridge (P/N WAT043380) in 10, 3 mLaliquots. After the addition of all loading solvent, two 3 mL aliquotsof 50 mM aqueous triethylammonium acetate (pH 7) containing 40%acetonitrile was loaded as a first wash. Two 3 mL aliquots of 50 mMaqueous triethylammonium acetate (pH 7) containing 60% acetonitrile wasthen loaded as a second wash. Finally, a single 3 mL aliquot ofacetonitrile was used to elute material remaining on the column. Theeluent of each aliquot was collected individually and analyzed by HPLCfor purity. The aliquots were vacuum evaporated and the mass of eachdetermined. Fractions of suitable purity were redissolved in DCM, thefractions were combined and precipitated in hexane. Yield 0.232 g.

Example 7

[0175] General Procedure for N-Terminal Labeling of Support Bound PNAwith Fluorescein-O-Linker

[0176] For N-terminal fluorescein labeling, the amino terminalfluorenylmethoxycarbonyl (Fmoc) group of several of the fully assembledPNA oligomers was removed by piperidine treatment and the resin waswashed and dried under vacuum. The resin was then treated for 20-30minutes with approximately 300 μL of a solution containing 0.07 MFluorescein-O-Linker, 0.06 M (HATU), 0.067 M DIEA and 0.1 M2,6-lutidine. After treatment the resin was washed and dried under highvacuum. The PNA oligomer was then cleaved, deprotected and purified asdescribed below.

Example 8

[0177] General Procedure for Labeling of Support Bound PNA with5(6)Carboxyfluorescein-NHS

[0178] This method was used as an alternative to the procedure describedin Example 7, for labeling PNAs with 5(6)-carboxyfluorescein. Thisprocedure requires that the N-terminus of the PNA oligomer be reactedwith Fmoc-8-amino-3,6-dioxaoctanoic acid prior to performing thelabeling reaction so that equivalent PNA constructs are prepared. Theamino terminal fluorenylmethoxycarbonyl (Fmoc) group of the fullyassembled PNA oligomer was removed by piperidine treatment and thesynthesis support was washed and dried under vacuum. The synthesissupport was then treated for 4-5 hours at 37° C. with approximately 300μL of a solution containing 0.1M 5(6)carboxyfluorescein-NHS (MolecularProbes, P/N C-1311), 0.3M DIEA and 0.3M 2,6-lutidine. After treatmentthe synthesis support was washed and dried under high vacuum. The PNAoligomer was then cleaved, deprotected and purified as described below.

[0179] More preferably, the synthesis support was then treated for 2-5hours at 30-37° C. with approximately 250 μL of a solution containing0.08M 5(6)carboxyfluorescein-NHS, 0.24M DIEA and 0.24M 2,6-lutidine.

Example 9

[0180] General Procedure for Labeling of Support Bound PNA with5(6)Carboxyfluorescein

[0181] After proper reaction with linkers and removal of the terminalamine protecting group, the resin was treated with 250 μL of a solutioncontaining 0.5M 5(6)carboxyfluorescein, 0.5MN,N′-diisopropylcarbodiimide, 0.5M 1-hydroxy-7-azabenzotriazole (HOAt)in DMF (See: Weberet al., Bioorganic & Medicinal Chemistry Letters, 8:597-600 (1998). After treatment the synthesis support was washed anddried under high vacuum. The PNA oligomer was then cleaved, deprotectedand purified as described below.

[0182] Note on Fluorescein Labeling: The fluorescein labeled PNAsdescribed herein were prepared using several different procedures. Thedifferent procedures have evolved to optimize fluorescein labelingconditions. At this time we prefer to use the procedure of Weber et al.for most fluorescein labeling operations.

Example 10

[0183] General Procedure for Dabcyl Labeling of the N-ε-Amino Group ofSupport Bound L-Lysine

[0184] This procedure was used as an alternative to using theprederivatized support when preparing dabcylated PNAs. This procedurehas the advantage that the lysine moiety (and therefore the attacheddabcyl moiety) may be placed at any location in the polymer includingwithin the probing nucleobase sequence.

[0185] The resin (still in the synthesis column) was treated with 10 mLof a solution containing 1% trifluoroacetic acid, 5% triisopropylsilane(TIS) in dichloromethane by passing the solution through the column overa period of approximately 15 minutes. After treatment, the synthesissupport was washed with DMF. Prior to treatment with labeling reagentthe support was neutralized by treatment with approximately 10 mL of asolution containing 5% diisopropylethylamine in DMF. After treatment,the support was treated with Dabcyl-NHS (as a substitute for5(6)carboxyfluorescein-NHS in the procedure) essentially as described inExample 8.

[0186] Note: This procedure was only performed on PNA prepared usingFmoc-PAL-PEG/PS (PerSeptive P/N GEN913384). It was not performed withthe more acid labile Fmoc-XAL-PEG/PS (PerSeptive P/N GEN913394).

Example 11

[0187] General Procedure for Cleavage, Deprotection and Purification

[0188] The synthesis support (Fmoc-PAL-PEG/PS; P/N GEN913384) wasremoved from the synthesis cartridge, transferred to a Ultrafree spincartridge (Millipore Corp., P/N SE3P230J3) and treated with a solutionof TFA/m-cresol (either of 7/3 or 8/2 (preferred)) for 1-3 hours. Thesolution was spun through the support bed and again the support wastreated with a solution of TFA/m-cresol for 1-3 hours. The solution wasagain spun through the support bed. The combined eluents (TFA/m-cresol)were then precipitated by addition of approximately 1 mL of diethylether. The precipitate was pelletized by centrifugation. The pellet wasthen resuspended in ethyl ether and pelletized two additional times. Thedried pellet was then resuspended in 20% aqueous acetonitrile (ACN)containing 0.1% TFA (additional ACN was added as necessary to dissolvethe pellet). The product was analyzed and purified using reversed phasechromatographic methods.

[0189] Note: Several PNAs were prepared using new productFmoc-XAL-PEG/PS synthesis support (P/N GEN 913394) available fromPerSeptive Biosystems, Inc. This support has the advantage that,the PNAcan be removed more rapidly and under more mildly acid conditions. ForPNAs prepared with Fmoc-XAL-PEG/PS the support was treated as describedabove except that a solution of TFA/m-cresol 9/1 was generally used fora period of 10-15 minutes (2×).

Experiment 12

[0190] Analysis and Purification of PNA Oligomers

[0191] All PNA probes were analyzed and purified by reversed phase HPLC.Probe composition was confirmed by comparison with theoreticalcalculated masses. The crude products for PNA probes P3 and P4 (Table 5)were prepurified using anion exchange chromatography prior to reversedphase HPLC purification. Anion exchange chromatography generallyimproved the purity level to better than 70 percent. Sephadex (PharmaciaBiotech) was used as the stationary phase and the mobile phase was 10 mMsodium hydroxide with a sodium chloride gradient.

[0192] HPLC Procedures:

[0193] Generally, two different high performance liquid chromatography(HPLC) gradients were used to analyze and purify the PNA oligomers(Gradients A & B). Preparative purifications were scaled based on theanalytical analysis conditions described in Gradients A & B. Gradient Bwas developed because initial purification using standard gradients(Gradient A) proved to be less than satisfactory. The experimentalconditions are as described below except that some attempts were made toimprove purifications by the addition of 20% formamide to the runningbuffers during some of the purifications. This procedure was abandonedsince it did not appear to produce any beneficial results. Curiouslyhowever, careful review of the data suggested that the HPLC artifactspreviously thought to correlate with the structure of certain probes(See: Provisional Patent Application No. 60/063,283 filed on Oct. 27,1997) was also found to correlate with the presence of formamide duringthe purification. Therefore, no correlation is now believed to existbetween structure of the PNA probe and the HPLC profiles observed forthe purified oligomers.

[0194] Gradients A & B

[0195] Buffer A=0.1% TFA in water.

[0196] Buffer B=0.1% TFA in acetonitrile.

[0197] Flow Rate: 0.2 mL/min.

[0198] Column Temperature: 60° C.

[0199] Instrument: Waters 2690 Alliance: Control by Waters MillenniumSoftware

[0200] Stationary Phase: Waters Delta Pak C18, 300 Å, 5 μm, 2×150 mm(P/N WAT023650)

[0201] Detection at 260 nm

[0202] Time (min.) Percent Buffer A Percent Buffer B Curve GradientProfile A 0.00 100 0 0 4.00 100 0 6 22.00 80 20 6 38.00 40 60 6 40.00 2080 11 Gradient Profile B 0.00 90 10 0 40.00 60 40 6 50.00 20 80 6

[0203] Mass analysis:

[0204] Samples were analyzed using a linear Voyager Delayed ExtractionMatrix Assisted Laser Desorption Ionization-Time Of Flight (DEMALDI-TOF) Mass spectrometer (PerSeptive Biosystems, Inc.). Sinipinicacid was used as the sample matrix and also used as one point forcalibration of the mass axis. Bovine insulin was used as an internalcalibration standard for the second calibration point of the mass axis.

[0205] Samples were generally prepared for analysis by first preparing asolution of sinipinic acid at a concentration of 10 mg/mL in a 1:2mixture of acetonitrile and 0.1% aqueous trifluoroacetic acid. Next, aninsulin solution was prepared by dissolving 1 mg of bovine insulin(Sigma) in 0.1% aqueous trifluoroacetic acid. Finally, an insulin/matrixsolution was then prepared by mixing 9 parts of the sinipHiic acidsolution to 1 part of the bovine insulin solution. Samples were preparedfor analysis by spotting 1 μL of the insulin/matrix solution followed byspotting 1 μL of diluted sample (approximately 0.1 to 1 OD per mL) ontothe mass spectrometer target. - The instrument target was allowed to drybefore being inserted into the mass spectrometer.

[0206] Tables of PNA Oligomers Prepared for Study TABLE 1 ProbesPrepared To Evaluate PNA Molecular Beacons (Hairpins) Probe Desc. CODE¹PNA Probe Sequence N-terminal Arm Forming Segments 0.001 5205Flu-O-TGG-AGO-OAC-GCC-ACC-AGC-TCC-AK(dabcvl)-NH₂ 0.007 5105Flu-O-TGG-AGO-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ 0.010 5005Flu-O-TGG-AGA-CGC-CAC-CAG-CTC-CAK(dabcyl)-NH₂ 0.002 3203Flu-O-TGG-OOA-CGC-CAC-CAG-CTC-CAK(dabcyl)-NH₂ 0.008 3103Flu-O-TGG-OAC-GCC-ACC-AGC-TCC-AK(dabcyl)-NH₂ 0.009 4004²Flu-O-TGG-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ C-terminal Arm FormingSegments 0.018 7027 Flu-O-ACG-CCA-CCA-GCT-CCA-OO-GTG-GCG-T-K(dabcvl)-NH₂0.011A 5025 Flu-O-ACG-CCA-CCA-GCT-CCA-OOG-GCG-TK(dabcyl)-NH₂ 0.006 3023Flu-O-ACG-CCA-CCA-GCT-CCA-OOC-GTK(dabcyl)-NH₂ Probing Sequence Externalto the Arm Sequences 0.017 5115Flu-O-TAG-CAO-ACG-CCA-CCA-GCT-CCA-OTG-CTA-K(dabcvl)-NH₂ 0.005 3113Flu-O-TAG-O-ACG-CCA-CCA-GCT-CCA-O-CTA-K(dabcyl)-NH₂ Control Probes; NoArm Forming Segments 0.003 0000 Flu-O-ACG-CCA-CCA-GCT-CCA-K(dabcvl)-NH₂0.004 0110 Flu-OO-ACG-CCA-CCA-GCT-CCA-OK(dabcyl)-NH₂ # number offlexible linker units which link the N-terminal arm to the probingnucleobase sequence. The third digit in the CODE represents the numberof flexible linker units which link the C-terminal arm to the probingnucleobase sequence. The fourth digit in the CODE represents the lengthof the C-terminal arm segment which is complementary to the N-terminalarm segment. Consequently, the CODE can be used to visually compare thegeneral structure of the different PNA oligomers listed in # Table 1.

Example 13

[0207] Synthesis of DNA Oligonucleotides Prepared for Study

[0208] For this study, biotin labeled DNA oligonucleotides suitable asnucleic acids comprising a target sequence which are complementary tothe PNA probing nucleobase sequence of the k-ras PNA probes were eithersynthesized using commercially available reagents and instrumentation orobtained from commercial vendors. All DNAs were purified by conventionalmethods. The sequences of the DNA oligonucleotides prepared for Examples14-18 and 20-22 are illustrated in Table 2. Methods and compositions forthe synthesis and purification of synthetic DNAs are well known to thoseof ordinary skill in the art. TABLE 2 DNA Targets Descrip- tion TargetDNA Sequence wt k-ras Biotin- GTG-GTA-GTT-GGA-GCT- G GT- Seq.Id.No.1GGC-GTA mu k-ras Biotin- GTG-GTA-GTT-GGA-GCT- T GT- Seq.Id.No.2 GGC-GTAUniv. Biotin- Comp. ACT-CCT-ACG-GGA-GGC-AGC Seq.Id.No.3

[0209] Initial Experimental Analysis Of PNA Molecular Beacons

[0210] In the initial experiments using a fluorescence detectioninstrument and PNA oligomers 0.001 and 0.002, it was determined that thePNA constructs have very little intrinsic fluorescence at roomtemperature. However, upon hybridization of either PNA oligomer to itscomplementary target sequence, an increasing fluorescent signal wasobserved.

Example 14

[0211] Hybridization Experiments

[0212] Amounts of PNA oligomer and target DNA used in this experimentare recorded in Table 3. The PNA oligomer and/or the target DNA wasmixed in 20 μl of Hybridization Buffer (50 mM Tris-HCl, pH 8.3; 100 mMNaCl) and heated to 95° C. for 10 minutes. After cooling slowly to roomtemperature, the mixture was diluted to a total volume of 4 mL (vol.needed in cuvette for measurement). Control samples containingHybridization Buffer (Hyb. Buffer) and the individual DNA or PNAoligomers were also examined under identical conditions. Additionally, afluorescein labeled PNA without quencher or arm forming segment wasincluded (Flu-OO-ACG-CCA-CCA-GCT-CC A-NH₂; “F-PNA”). The experimentalmeasurements which were recorded are reproduced in Table 3.

[0213] With reference to Table 3, there was a low backgroundfluorescence from the individual components of the test system (e.g.hybridization buffer, single stranded DNA, and PNA oligomers). Whentarget DNA and PNA oligomer was mixed and allowed to hybridize, asignificant increase in fluorescent signal was detected. Moreover, theintensity of fluorescent signal varied as the relative concentrations ofthe target DNA and PNA oligomer was altered. Consequently, the datademonstrates that hybridization of the PNA oligomers to thecomplementary DNA target generated very intense fluorescent signal.

[0214] The signal obtained using PNA oligomer 0.002 (3 bp. stem) wasbetween 29 and 83% higher than the signal observed for the PNA oligomer0.001 (5 bp. stem) (Compare: data in rows 2, 7 and 12 with data in rows,1, 6 and 11). However, as demonstrated by the greater fluorescentintensity observed for the control probe (F-PNA), the presence of thequenching moiety attached to the PNA oligomer results in a significantquenching effect (Compare: data in rows 3, 13 and 16 with data in rows1-2, 6-7 and 11-12). Because the fluorescence of the control probe F-PNAwas so intense, it was diluted to obtain fluorescent signal which wascomparable in intensity to the data obtained using a PNA oligomer havingan linked dabcyl quencher moiety. Specifically, the signal obtained withthe fluorescein labeled control PNA oligomer (F-PNA) was approximatelytwo to three times the greatest intensity of the signal generated fromthe PNA oligomers containing a quencher moiety. TABLE 3 Ex. L(490) RowNo. Assay Components pmol 521 nm 1 wt k-ras DNA/PNA .001 125/25  300 2wt k-ras DNA/PNA .002 125/25  447 3 wt k-ras DNA/F-PNA 12.5/2.5  87 4PNA .001 25 1 5 PNA .002 25 1 6 wt k-ras DNA/PNA .001 25/25 189 7 wtk-ras DNA/PNA .002 25/25 345 8 wt k-ras DNA/F-PNA 12.5/12.5 423 9 PNA.001 25 1 10 PNA .002 25 1 11 wt k-ras DNA/PNA .001  25/125 353 12 wtk-ras DNA/PNA .002  25/125 455 13 wt k-ras DNA/F-PNA  2.5/12.5 373 14PNA .001 125  25 15 PNA .002 125  38 16 wt k-ras DNA/F-PNA 12.5/12.5 42317 wt k-ras DNA 125  −3 18 Hybridization Buffer — −5

Example 15

[0215] Titration of PNA Oligomer with Nucleic Acid Target

[0216] In another experiment, 50 pmol of PNA oligomers 0.001 and 0.002were mixed with differing amounts of nucleic acid target (0-200 pmol) ina total volume of 20 μl of Hyb. Buffer. The mixtures were then heated to95° C. for 10 minutes and cooled slowly to ambient temperature. Thesamples were diluted into a total volume of 4 mL and excitation/emissionat 493/521.6 nm was recorded using a RF-5000 spectrofluorophotometer(Shimadzu). Results are illustrated graphically in FIG. 1.

[0217] With reference to FIG. 1, the fluorescent signal generated fromthe sample continuously increased with the addition of target sequenceuntil a concentration of 40-60 pmol was present (50 pmol PNA oligomerwas used in the assay). There was no significant increase in fluorescentsignal as the amount of target sequence was increased between 60-200pmol. Consequently, the data demonstrates that the signal generated inproportional to the amount of target sequence added, thereby indicatingthat the production of the signal was caused by the hybridization of thePNA oligomer to the target nucleic acid.

Example 16

[0218] Kinetics of Hybridization for PNA Molecular Beacons

[0219] In this experiment, 100 pmol (5 μL of 20 pmole/μL) of wt k-rasDNA (ssDNA oligonucleotide) was mixed with 4 mL Hybridization Buffer ina cuvette and adjusted to ambient temperature. Next, 50 pmol (2.5 μL of20 pmole/μL) of PNA oligomer 0.002, was added and theexcitation/emission at 493/521.6 nm was recorded RF-5000spectrofluorophotometer (Shimadzu). The data obtained is graphicallyillustrated in FIG. 2.

[0220] With reference to FIG. 2, the results demonstrate that the PNAMolecular Beacon hybridizes to the target DNA present in the sample tothereby generate a fluorescent signal with measurable kinetics. Thegeneration of fluorescent signal occurred with an initial rate of 7.2relative light units (rlu)/minute. After 120 minutes of hybridizationthe signal was 595 rlu. The kinetic profile of the increase influorescent intensity is strongly indicative of hybridization of the PNAoligomer to the target nucleic acid.

Example 17

[0221] Hybridization Related to the Composition of the DNA Target

[0222] In this experiment, 50 pmol of either wt k-ras or mu k-ras DNA,in hybridization buffer, was mixed with 50 pmol PNA Molecular Beacon0.001 in hybridization buffer. As a control, 50 pmol of a totallyunrelated target DNA oligonucleotide, mixed with 50 pmol of PNAMolecular Beacon 0.001, was also examined (Univ. Comp., See: Table 2).Excitation/emission at 493/521.6 nm was recorded was recorded RF-5000spectrofluorophotometer (Shimadzu). The results are presentedgraphically in FIG. 3.

[0223] With reference to FIG. 3, the increasing fluorescent intensityobserved when mixing PNA 0.001 and the complementary target sequencedemonstrates that the PNA Molecular Beacon hybridized to its perfectcomplement wt k-ras DNA. The rate of fluorescent signal generation andthe maximal fluorescent signal generated was significantly lower for thesample when the non-complementary mu k-ras DNA (having a single pointmutation as compared with the wild type target) was substituted in theassay. Furthermore, mixing a totally unrelated nucleic acid sequence(Univ. Comp) with PNA Molecular Beacon 0.001 did not produce anyfluorescent signal, even after heating the sample to 95° C.Consequently, the data strongly demonstrates that the generation of thefluorescent signal in the assay was directly related to the sequencespecific interactions between the PNA Molecular Beacon and the nucleicacid present in the sample.

Example 18

[0224] Effect of Blocking Probes on Sequence Discrimination

[0225] This experiment was designed to increase discrimination of thePNA oligomer 0.001 by introducing some unlabeled “blocking PNA” into thehybridization mixture. This “blocking PNA” has a sequence complementaryto the mu k-ras DNA and should, therefore, effectively compete with thelabeled PNA oligomer 0.001 for binding to the non-target mu k-ras DNA.The unlabeled “blocking PNA” had the sequence:

[0226] Blocking PNA: H-ACG-CCA-CAA-GCT-CCA-NH₂

[0227] In this experiment, 50 pmol of wt k-ras or mu k-ras DNA was mixedwith 50 pmol PNA oligomer 0.001. To one pair of reactions was added 500pmol “blocking PNA” (data in Table 4, row 2). The samples were heated to95° C. and then cooled slowly to RT. Excitation/emission at 493/521.6 nmwere recorded. The results obtained are reproduced in Table 4.

[0228] With reference to Table 4, in the absence of blocker PNA there isa 2 fold difference (discrimination factor) between the fluorescentsignal generated when the PNA oligomer hybridizes to the perfectlycomplementary wt k-ras DNA as compared with signal generated uponhybridization to non-complementary mutant k-ras DNA (single pointmutation). Addition of “blocking PNA” increases the discrimination ofthe assay by approximately 10 fold (Compare data in Table 4, thediscrimination factor (DF) is increased to 19.6 from 2). This increasein discrimination occurred despite an approximately 50% reduction in thesignal generated by hybridization to the complementary wt k-ras DNA.However, as demonstrated by analysis of the data in Table 4,hybridization of the PNA probe to the non-complementary mu k-ras DNA waseffectively eliminated in the presence of the “blocking PNA”.Consequently, the data again strongly illustrates that the generation offluorescent signal was directly related to the sequence specifichybridization of the nucleic acid to the PNA oligomer. The data furtherdemonstrates the utility of using blocking probes to enhance sequencediscrimination in nucleic acid hybridization assays when utilizing PNAMolecular Beacons. TABLE 4 Row wt k-ras DNA/ mu k-ras DNA/ No.Conditions PNA .001 PNA .001 DF 1  0 pmol Blocking PNA 541 271 2 2 500pmol Blocking PNA 255 13 19.6

[0229] Conclusions from the Initial Experimental Data:

[0230] PNA Molecular Beacons can be prepared. They have low intrinsicfluorescent intensity until hybridized to a complementary orsubstantially complementary target nucleic acid. Non-specificinteractions can be eliminated, if desired, using a “blocking PNA”.Thus, we have demonstrated that it is possible to prepare functional PNAMolecular Beacons which exhibit a good specificity which can be furtherenhance by the application of “blocker probes”. Consequently, the PNAoligomer constructs investigated in these initial experimentsdemonstrate the effective use of PNA Molecular Beacons in probe-basedhybridization assays.

Example 19

[0231] Structural Analysis of PNA Hairpins and Multimers

[0232] The reference entitled “Hairpin-Forming Peptide Nucleic AcidOligomers”, Armitage et al., Biochemistry, 37: 9417-9425 (1998) isadmitted as prior art to this Example 19 only. It has been recentlyreported in the scientific literature that PNAs form hairpin structures(See: Armitage et al.). Using the hairpin forming PNA (referred to as“PNA1” in the reference (See: Scheme 3: col. 1, p. 9419), hereinafterreferred to as “PNAD”) described in the literature as a model, numerousPNA and analogous DNA oligomers were prepared and their propertiesexamined in order to obtain a basis for understanding the physicalbehavior of PNA hairpins and multimers. We anticipated this would allowus to better interpret the results reported in our priority application(U.S. Ser. No. 08/958532). The data presented in this Example 19demonstrates that PNA hairpins will form if designed to have long stems(e.g. 9 subunits). However PNA hairpins having shorter stems (e.g. 6subunits) do not form hairpins as readily as their DNA counterparts.Furthermore, applicants have observed that the formation and stabilityof a PNA hairpin is not substantially affected by the presence orabsence of magnesium or the ionic strength of the buffer as are DNAhairpins (See Tyagi et al., Nature Biotechnology, 14: 303-308 (1996) atp. 305, col. 1, lns 1-16). Nevertheless, PNA hairpins having stemduplexes of 9 subunits in length exhibit poor signal to noise ratios(less than 4 to 1) upon melting and, contrary to the findings ofArmitage et al., do not appear to substantially hybridize tocomplementary nucleic acid. Consequently, PNA hairpins which have longstems (e.g. 7 or greater subunits), do not appear to be ideally suitedfor the analysis of nucleic acids.

[0233] Materials and Methods:

[0234] Probes

[0235] PNAs were prepared and purified as described herein. Labeled andunlabeled DNA oligonucleotides were synthesized using commerciallyavailable reagents and instrumentation. Dabcylated DNAs were preparedusing the dabcyl synthesis support available from Glen Research (P/N20-5911) and other commercially available DNA reagents andinstrumentation. The Fluoredite phosphoramidite (PerSeptive Biosystems,Inc., P/N GEN080110) was used to label DNAs with 5(6)carboxyfluorescein.All DNAs were purified by conventional methods. The DNA and PNA probecompositions are presented ill Table 5. Tm data for DNA probes issummarized in Table 6 and the Tm data for PNA probes is summarized inTable 7. Tm data for both the melting “M” and the reannealing “R” ispresented in Table 6 and 7.

[0236] Preparation of Dilution Series of PNA and DNA Probes for TmAnalysis

[0237] Purified PNA probes were dissolved in 1:1 DMF/H₂O at 0.05 OD (260nm ) per 20 μL to prepare the PNA Probe Stock. Purified DNA probes weredissolved in 4:1 H₂O/acetonitrile at 0.05 OD (260 nm) per 20 ∥L toprepare the DNA Probe Stock. Based on calculated extinctioncoefficients, the appropriate amount of PNA Probe Stock or DNA ProbeStock was added to 5 ml of Tm Buffer (10 mM sodium phosphate, pH 7.0) toprepare a solution of approximately 7.5 μM of the one or two oligomersneeded to perform the Tm analysis of the unimolecular or bimolecularsystem. From this solution was taken 2.5 mL which was added to 2.5 mL ofTm buffer to thereby prepare the second concentration of a dilutionseries of Tm Samples. The remaining 2.5 mL of the first sample was usedfor Tm analysis. Serial dilution the samples in Tm Buffer was continuedin this fashion until 2.5 mL of Tm Samples at concentrations ofapproximately 7.5 μM, 3.75 μM, 1.87 μM, 0.94 μM and 0.468 μM (5 mL) wereprepared. A Tm analysis of these solutions was then performed asdescribed below.

[0238] Tm Analysis

[0239] 1. Tm Buffer:

[0240] The five Tm Samples of a dilution series of a particularunimolecular or bimolecular system to be analyzed were simultaneouslyexamined using a Cary 100 Bio UV-Visible Spectrophotometer (VarianInstruments) equipped with a 6×6 thermostatable multicell block runningWin UV Bio application software package. To a 10×10 UV cell (StarnaCells, P/N 21-Q-10) was added a 7.2 mm stir bar and the 2.5 mL of eachsample of the dilution series. The stirring accessory was used duringall analysis. All samples were thermally denatured and reannealed priorto data collection by having the instrument rapidly ramp the temperatureto a point above the melting temperature and then holding thattemperature for 5-10 minutes before returning to the startingtemperature. Data for both dissociation and reannealing was collectedand analyzed. The temperature range over which data was collected wasvaried in response to the expected Tm which was roughly determinedduring the premelt and prereannealing step. Regardless of thetemperature range, the temperature ramp rate for both dissociation andreannealing was always 0.5° C./min. The absorbance (260 nm, averagedover a 3 second collection) was plotted vs. the temperature of themulticell block.

[0241] 2. Tm Buffer and 1 mM MgCl₂:

[0242] After the Tm analysis was performed in Tm Buffer, to each cellwas added 0.5 μL of 5M MgCl₂ to thereby prepare a sample containing 1 mMMgCl₂. The dilution effect was considered to be negligible. The Tmanalysis was then performed again to determine whether the presence ofMgCl₂ had any effect on the Tm of the unimolecular or bimolecularsystem.

[0243] 3. Tm Buffer, 1 mM MgCl₂ and 100 mM NaCl:

[0244] After the Tm analysis was performed in Tm Buffer and 1 mM MgCl₂,to each cell was added 42 μL of saturated NaCl (approximately 6.11 M/L).The dilution effect was again considered to be negligible. The Tmanalysis was then performed again to determine whether the presence ofapproximately 100 mM NaCl had any effect on the Tm of the unimolecularor bimolecular system.

[0245] Results and Discussion:

[0246] With reference to Table 5, the sequences of complementary labeledPNA probes P1 and P2 are illustrated. These probes hybridize to form a 9subunit duplex identical to the stem duplex formed in PNA hairpins, P3and P4. Therefore, P1 and P2 form a bimolecular system for comparisonwith the unimolecular PNAs, P3 and P4. Probes P3 and P4 differ only inthat the subunits which form the loop of P4 have been replaced withflexible linkages in P3. Unlabeled versions of all four PNA probes havelikewise been prepared so as to understand the effects of labels on Tm.In Table 5, the unlabeled probes are designated with an “N” for nolabel.

[0247] Also prepared for comparison is the PNA probe “PNAD” whichArmitage et al. teach will form a hairpin. Applicants have also prepareda version of the PNAD probe which possess arm segments of 6 subunits(PNAD 6S) as compared with the 9 subunits self complementary armsegments of the PNAD probe. The PNA probe P5 is complementary to the DNAprobe D5B. This bimolecular complex was prepared to determine its Tmsince Armitage et al. teach that this short 12-mer nucleic acid sequencewill hybridize to the PNAD probe thereby opening the 9 subunit stem.TABLE 5 Sequence Probe Sequence ID Seq Id. No. Peptide Nucleic AcidProbes Flu-O-ATA-TAT-TGG-EE-NH₂ P1 Ac-O-ATA-TAT-TGG-EE-NH₂ P1NAc-EE-CCA-ATA-TAT-K(dabcyl)-NH₂ P2 Ac-EE-CCA-ATA-TAT-NH₂ P2NFlu-OEE-ATA-TAT-TGG-OO-CCA-ATA-TAT-EE-K(dabCyl)-NH₂ P3H₂N-OEE-ATA-TAT-TGG-OO-CCA-ATA-TAT-EEK-NH₂ P3NFlu-OEE-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-EE-K(dabCyl)-NH₂ P4H₂N-OEE-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-EEK-NH₂ P4NH₂N-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-KK-NH₂ PNADH₂N-TAT-TGG-CTG-ATC-CAA-TA-KK-NH₂ PNAD 6S H₂N-TTG-GCT-GAT-CCA-NH₂ P5Synthetic Oligodeoxynucleotides Flu-ATA-TAT-TGG-OH D1 Seq. Id. No. 4HO-ATA-TAT-TGG-OH D1N Seq. Id. No. 5 HO-CCA-ATA-TAT-(dabcyl) D2 Seq. Id.No. 6 HO-CCA-ATA-TAT-OH D2N Seq. Id. No. 7Flu-ATA-TAT-TGG-(spacer)-CCA-ATA-TAT-dabcyl D3 Seq. Id. No. 8HO-ATA-TAT-TGG-(spacer)-CCA-ATA-TAT-OH D3N Seq. Id. No. 9Flu-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-dabCyl D4 Seq. Id. No. 10HO-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-OH D4N Seq. Id. No. 11Bio-TGG-ATC-AGC-CAA-OH D5B Seq. Id. No. 12HO-ATA-TAT-TGG-ATC-AGC-CAA-TAT-AT-OH D6 Seq. Id. No. 13

[0248] With reference to Table 5, the sequences for the syntheticoligodeoxynucleotides prepared for examination are also illustrated.DNAs which were conceptually the most equivalent labeled and unlabeledversions of P1, P2, P3, P4, P1N, P2N, P3N and P4N were prepared forcomparison. As discussed, D5B is a complement to P5 and D6 is thecomplement to D4, P4 and PNAD. TABLE 6 UV Tm Analysis [1] [2] [3] [4][5] Probes (Conditions) M R M R M R M R M R D1N/D2N (Buf) <10 <10 <10<10 <10 <10 <10 <10 <10 <10 D1N/D2N (Buf, Mg) 17.1 16.8 14.6 14.8 14.013.4 11.4 11.7 <10 <10 D1N/D2N (Buf, Mg & Na) 22.3 21.7 20.7 18.9 19.018.2 15.0 15.0 14.2 13.4 D1/D2 (Buf) 15.0 14.8 13.5 13.3 12.0 12.9 11.59.9 <10 <10 D1/D2 (Buf, Mg) 24.1 23.8 22.0 21.8 21.0 20.9 19.5 19.9 16.517.9 D1/D2 (Buf, Mg & Na) 29.0 28.4 27.5 26.4 26.0 25.5 23.4 23.0 21.420.0 D3N (Buf) 40.0 40.5 40.5 40.5 40.5 40.5 40.5 41.2 40.5 41.2 D3N(Buf, Mg) 44.6 44.7 44.6 45.3 44.6 44.8 45.5 45.4 45.5 44.9 D3N (Buf, Mg& Na) 48.1 48.3 48.6 48.8 49.0 49.4 48.5 49.5 48.0 48.6 D3 (Buf) 43.743.6 44.3 44.1 44.3 44.6 43.9 44.0 43.9 44.0 D3 (Buf, Mg) 50.6 49.9 50.649.9 51.1 50.5 51.0 50.5 51.5 51.1 D3 (Buf, Mg & Na) 54.1 53.8 55.6 53.854.6 54.0 54.5 54.5 54.5 54.1 D4N (Buf) 41.6 41.7 42.1 42.2 42.5 42.342.5 42.8 42.0 43.4 D4N (Buf, Mg) 51.6 51.2 52.1 52.3 52.5 52.4 52.553.5 52.5 53.0 D4N (Buf, Mg & Na) 54.1 54.4 55.1 54.9 55.1 55.0 55.055.0 55.0 55.1 D4 (Buf) 45.2 43.4 45.6 44.9 45.6 45.0 45.5 44.5 45.544.1 D4 (Buf, Mg) 54.6 53.9 55.5 55.0 55.5 55.0 54.5 54.1 55.0 54.6 D4(Buf, Mg & Na) 57.1 56.5 57.5 57.5 58.0 57.5 58.5 57.6 58.0 57.6

[0249] With reference to Table 6, the Tm for the labeled and unlabeledbimolecular systems D1/D2 and D1N/D2N, respectively, are concentrationdependent as can be seen by the 8-10° C. difference between the most andleast concentrated samples where Tm data is available. The low andunmeasurable Tm values under conditions of low ionic strength areexpected for such a short nucleic acid duplex. However, there is aremarkable stabilizing effect of approximately 7° C. due to the presenceof the dabcyl/fluorescein labels as can be seen by comparison of the Tmvalues at all concentrations and under all conditions examined. This wasa very surprising result.

[0250] The Tm of labeled (D3 and D4) and unlabeled (D3N and D4N)unimolecular probes exhibited a concentration independent Tm.Consequently, the data indicates that these DNAs exist as hairpins insolution. As taught by Tyagi et al., the hairpin stem duplex of theseDNA probes is substantially stabilized by both the addition of magnesiumand an increase in ionic strength (See: Tyagi et al. NatureBiotechnology, 14: 303-308 (1996) at p. 305, lns. 1-16). Surprisinglythe stem duplex D4 (which contained a polynucleotide loop) was morestable under all conditions examined than was the stem duplex of D3which contained a flexible linkage. Also noteworthy is the stabilizingeffect attributable to the fluorescein/dabcyl pair. For D4 thestabilizing effect is approximately 3-4° C. whereas it is approximately5-6° C. for D3. Thus, the data indicates that as the Tm of the stemduplex increases there is less of a stabilizing effect attributable tothe dabcyl/fluorescein interactions. Nevertheless, this observation isvery surprising and suggests that the interaction between the dyes isvery strong. TABLE 7 UV Tm Analysis [1] [2] [3] [4] [5] Probes(Conditions) M R M R M R M R M R P1N/P2N (Buf) 57.1 56.5 55.0 54.6 53.052.6 51.5 50.6 48.5 48.7 P1N/P2N (Buf, Mg) 57.1 56.4 55.1 54.5 53.0 53.051.0 50.5 49.0 48.6 P1N/P2N (Buf, Mg & Na) 57.6 57.0 55.4 55.1 53.5 53.152.0 51.1 49.5 49.2 P1/P2 (Buf) 61.1 60.4 59.1 58.5 57.0 57.0 55.5 54.551.5 52.6 P1/P2 (Buf, Mg) 61.1 60.4 59.1 59.0 57.5 57.0 55.5 55.0 52.553.1 P1/P2 (Buf, Mg & Na) 61.5 60.9 59.6 59.5 58.0 57.0 56.0 55.5 54.053.6 P3N (Buf) 82.6 82.3 82.5 82.4 83.0 82.4 83.0 82.5 83.0 82.6 P3N(Buf, Mg) 83.1 82.4 83.1 81.9 83.0 82.5 83.0 83.1 83.0 82.6 P3N (Buf, Mg& Na) 83.1 82.9 83.1 81.4 83.5 82.9 83.5 83.0 83.5 83.1 P3 (Buf) 82.181.9 82.6 82.0 82.6 82.0 83.0 82.6 82.0 81.6 P3 (Buf, Mg) 82.1 81.8 82.181.8 82.5 82.4 82.5 82.0 83.0 83.5 P3 (Buf, Mg & Na) 83.1 82.9 83.5 83.084.0 83.0 83.5 83.0 83.5 nd P4N (Buf) 81.6 81.4 82.1 81.5 82.1 81.5 82.581.1 82.0 80.1 P4N (Buf, Mg) 81.6 81.3 81.6 81.3 82.1 81.4 82.0 nd 82.081.1 P4N (Buf, Mg & Na) 82.1 82.3 82.1 82.3 82.5 nd 82.0 82.0 84.0 81.6P4 (Buf) 81.6 80.9 81.6 81.4 82.1 81.5 82.0 81.5 81.5 81.1 P4 (Buf, Mg)81.6 81.0 81.6 81.5 82.0 81.5 82.0 81.1 82.0 81.6 P4 (Buf, Mg & Na) 82.681.8 82.6 82.4 82.6 82.5 82.5 82.5 83.0 82.1 PNAD (Buf) 81.1 80.4 81.181.0 82.0 81.0 81.5 81.5 81.5 81.1 PNAD (Buf, Mg) 80.6 80.3 81.1 80.481.1 80.4 80.5 81.0 80.5 80.5 PNAD (Buf, Mg & Na) 80.6 80.4 81.5 nd 81.081.4 81.5 80.5 82.1 80.6 PNAD 6S (Buf) 75.0 74.6 75.0 74.1 74.5 73.573.5 65.5 67.5 57.5

[0251] With reference to Table 7, Tm data for the PNA constructs ispresented. The Tm values for the PNAs are substantially higher than forthe comparable DNAs. Both the labeled (P1/P2) and unlabeled (P1N/P2N)bimolecular systems exhibited Tms which were concentration dependent asis evident by the 8-10° C. difference in Tm between the most and leastconcentrated samples. Again there was a increase of approximately 3-4°C. in Tm which was attributable to the presence of thefluorescein/dabcyl moieties. Though clearly dependent uponconcentration, the stability of the duplexes were not substantiallyaffected by the presence or absence of magnesium or the ionic strengthof the buffer since there was no substantial difference in Tm under anyof the three conditions examined. Most importantly no substantialhysteresis was observed in the analysis of labeled or unlabeled PNAbimolecular systems even at the lowest concentration examined. The lackof hysteresis indicates that the duplex reforms readily.

[0252] For comparison, fluorescence analysis of the least concentratedsample of the P1 /P2 bimolecular system was performed. The leastconcentrated sample (sample at [5] which had 1 mM MgCl₂ and 100 mM NaCl)was analyzed for fluorescence essentially as described in Example 20,below, except that excitation was at 415 nm and emission was recorded at520 nm. Normalized data for fluorescence vs. temperature and absorbancevs. temperature are overlaid in FIG. 4A. Though the shape of the curvesis similar the data is not superimposible. A similar result was observedwhen the absorbance vs. temperature and fluorescence vs. temperaturedata for the D1/D2 system was overlaid (data not shown). The structuralbasis for this lack of superimposibility is not known but appears to beconsistent for the bimolecular systems.

[0253] Both the labeled and unlabeled versions of P3 and P4 exhibited aconcentration independent Tm. Consequently, the data indicates thatthese PNAs form hairpins in solution. Likewise, the probe PNAD also wasconfirmed to exhibit a concentration independent Tm of approximately81-82° C., as had been reported by Amitage et al. The data clearlydemonstrates that the stem duplex of a PNA hairpin is not substantiallyaffected by the presence or absence of magnesium or the increase inionic strength since the Tm for the probes are the same without regardto the buffer composition in which the Tm analysis was performed.Curiously there was no substantial difference in the Tm of labeled ascompared with unlabeled probes. However, it is believed that the Tm ofthese duplexes is so high that the fluorescein/dabcyl interactionscannot be maintained.

[0254] As with the P1/P2 and D1/D2 bimolecular systems, fluorescence vs.temperature analysis of the least concentrated samples (each probe at[5] which had 1 mM MgCl₂ and 100 mM NaCl) of both probes P3 and P4 wereperformed. With reference to FIGS. 4B and 4C, normalized fluorescencevs. temperature and absorbance vs. temperature data are overlaid for P3and P4, respectively. Unlike the bimolecular system, the fluorescencevs. temperature and absorbance vs. temperature data for both P3 and P4are superimposible. Data was also collected for the D3 and D4unimolecular probes. The data for these unimolecular probes was alsofound to be highly superimposible (data not shown) thereby indicatingthey result from the same physical transition of the probe. Taken as awhole, the excellent correlation between the fluorescence vs.temperature and absorbance vs. temperature data in both the DNA and PNAunimolecular systems strongly indicates that increases in absorbance andfluorescence occur as result of a helix to coil transition.

[0255] Using the data obtained from melting and reannealing of D1/D2,D3, D4, P1/P2, P3 and P4 as described above, the difference between thelowest (helix) and highest (coil) fluorescence intensities recorded werecalculated to determine the signal to noise value for each probe. Thiswas intended to give an estimate of the potential increase in signalwhich could be expected in a hybridization assay wherein the stern ofthe probe was opened. In FIG. 5, the fluorescence signal to noise ratiosfor melting and reannealing the PNA and DNA bimolecular and unimolecularsystems is presented. Most striking is the significantly lower signal tonoise ratio for all the PNA systems as compared with the DNA systems.The low signal to noise ratio is consistent with the data presented byArmitage et al. though it is not clear that the labeled probes ofArmitage et al. form hairpins. Nevertheless, the low signal to noiseratios for the PNA probes comprising long self-complementary armsegments suggests that these constructs are not optimal for analysis ofnucleic acids.

[0256] Though the Tm of labeled and unlabeled hairpins having anidentical 9 bp stem duplex where all very similar (approximately 81-83°C.), normalized data presented in FIG. 6 demonstrates that severalfactors can influence thermodynamic parameters of the stem duplex. InFIG. 6, normalized absorbance vs. temperature data for melting of probesP3N, P4N and PNAD (each probe at [1]) is graphically illustrated. Asthese probes were all unlabeled and comprised the same nucleobasesequence there were directly comparable. Probe P3N which comprises aflexible linkage which links the two arms which form the stem duplexexhibited the most cooperative sigmoidal transition. Surprisingly, thesolubility enhanced probe, P4, exhibited only a slightly lesscooperative transition as compared with probe P3. The probe PNADexhibited the least cooperative sigmoidal transition.

[0257] The shape of the sigmoidal transition evident in absorbance vs.temperature plots is a function of the properties of the duplex. Sharpcooperative transitions are expected for the more thermodynamicallystable duplexes whereas sloping sigmoidal transitions are expected wherethe duplex is less thermodynamically stable. The flexible linkage in P3was expected to stabilize the duplex so the sharp transition observedwas expected. The substantial difference between probe P4 and PNADhowever was surprising and can only be attributed to the presence of thesolubility enhancers.

[0258] The data presented in FIG. 6 lead us to believe that althoughprobe PNAD was a hairpin, it appeared to be borderline in stability.Therefore we theorized that a probe with shorter arm segments (e.g. 6subunits in length) might not exist predominately as a hairpin sincePNAs are known to be organized in solution (See: Dueholm et al., New J.Chem., 21: 19-31 (1997) at p. 27, col. 2, lns. 6-30). With reference toTable 7, the data presented for probe PNAD 6S, which is designed with asix subunit self-complementary arm segment, as compared with the 9subunit arm segments of the PNAD probe, confirms that the probe does notexist primarily as a hairpin since the Tm is concentration dependent.Moreover, there are two inflection points in the reannealing curve (datanot shown) at low concentrations (samples at [4] and [5])) which isindicative of the existence of both hairpin and multimer formation.Consequently, the data indicates that PNAs having arm segments of 6 orless subunits, and no flexible linkage groups, do not exist primarily ashairpins.

[0259] The bimolecular duplex, P5/D5B was also analyzed to determine itsTm. The data obtained by applicants indicated that the most concentratedsample (approximately 7.5 μM) had a Tm of 71° C. At half concentrationthe Tm was approximately 70° C. and at one quarter concentration the Tmwas approximately 68.5° C.

[0260] The DNA probe D5B was complementary to only a portion of PNAprobes, P3, P4 and PNAD. The DNA probe, D6, was perfectly complementaryto P4 and PNAD and a portion of P3. Hybridization assays were performedto determine whether probes D5B or D6 would hybridize with probes P3 orP4, thereby opening the hairpin stem duplex and generating fluorescentsignal. Hybridizations were performed essentially as described inExample 21, below except that the DNA target was in excess. The dataobtained indicated that very little hybridization occurred after 30minutes. As these are the most favored duplexes given the perfectcomplementary of the probes and targets, the lack of detectable signalin the hybridization reaction indicates that little or no hybridizationoccurs. Consequently, the data suggests that probes having long stems(e.g. 7-10) and no flexible linkages are not optimal for analyzingsamples for a nucleic acid target since they do not produce detectablesignal.

[0261] These hybridization results should be expected since the Tm ofthe PNA/DNA duplex should be lower than the Tm of the hairpin stemduplex. For example, the Tm of the perfect complement P5/D5B isapproximately 71° C. at concentrations much higher than the effectiveconcentration of reactants in the hybridization reaction whereas theconcentration independent Tm of the hairpin stem duplex is 81-82° C.Thus, it is not reasonable to expect that the short DNA probe, D5B, willsubstantially hybridize to P4 and open the more stable hairpin stemduplex.

[0262] In summary, the data presented in this Example 19 demonstratesthat PNAs with long self-complementary arm segments (e.g. 9 subunits)and no flexible linkages form stable hairpins while those having shorterarm segments (e.g. 6 subunits ) and no flexible linkages are likely toexist in-both the hairpin and multimer state. When hairpins are formed,the Tm of the stem duplex is substantially independent of the presenceor absence of magnesium and the ionic strength of the buffer.Unfortunately, the data compiled by applicants indicates that labeledprobes most likely to form hairpins, because they possess longerself-complementary arm segments (but do not comprise flexible linkages),exhibit very poor signal to noise ratios in both hybridization andthermal melting experiments. This data suggests that these probes arenot well suited for use in the detection of nucleic acid targets. Themost surprising result was the substantial stabilizing effectattributable to the dabcyl/fluorescein interactions. Such stronginteractions may explain why quenching occurs regardless of lack ofsubstantial spectral overlap between dabcyl and fluorescein (i.e. bynon-FRET).

[0263] Detailed Analysis of PNA Oligomers Prepared for Study

[0264] Experiments 20-22 were performed to generate comparative data onthe PNA oligomers in Table 1 so that preferred configurations of PNAMolecular Beacons could be determined. Generally the data indicates thatthe insertion of flexible linkages within the probes improves signal tonoise ratios particularly when the flexible linkage is inserted at theN-terminus of the probe to thereby link an arm segment to the probingnucleobase sequence. The data also indicates that probes with shorterarm segments also generally exhibit a more favorable signal to noiseratio. Several of the probes exhibited signal to noise ratios which werecomparable with nucleic acid constructs which are self-indicating (e.g.a nucleic acid Molecular Beacon). Therefore, the PNA Molecular Beaconsof this invention are useful for detecting nucleic acid targets insamples of interest. However, the data is inconclusive with regard towhether or not any of the PNA probes listed in Table 1 exist primarilyas hairpins. Furthermore, the data indicates that, under the sameexperimental conditions, the properties of the probes listed in Table 1vary substantially from probe to probe under the conditions examined.Several of the results are not well understood. Thus, it has not beenpossible to characterize the PNA probes listed in Table 1.

Example 20

[0265] Analysis of Fluorescent Thermal Profiles:

[0266] General Experimental Procedure:

[0267] Fluorescent measurements were taken using a RF-5000spectrofluorophotometer (Shimadzu) fitted with a water jacketed cellholder (P/N 206-15439, Shimadzu) using a 1.6 mL, 10 mm path length cuvet(Starna Cells, Inc.). Cuvet temperature was modulated using acirculating water bath (Neslab). The temperature of the cuvet contentswas monitored directly using a thermocouple probe (Barnant; model No.600-0000) which was inserted below liquid level by passing the probe tipthrough the cap on the cuvet (custom manufacture).

[0268] Stock solution of HPLC purified PNA oligomer was prepared bydissolving the PNA in 50% aqueous N,N′-dimethylformamide (DMF). Fromeach PNA stock was prepared a solution of PNA oligomer, each at aconcentration of 10 pmol in 1.6 mL of Hyb. Buffer (50 mM Tris. HCl pH8.3 and 100 mM NaCl) by serial dilution of purified PNA stock with Hyb.Buffer.

[0269] Samples were exited at 493 nm and the fluorescence measured at521 nm. Data points were collected at numerous temperatures as the cuvetwas heated and then again measured as the cuvet was allowed to cool.Generally, the bath temperature was sequentially increased by 5° C. andthen allowed to equilibrate before each data point was recorded.Similarly, to generate the cooling profile, the bath temperature wassequentially decreased by 5° C. and then allowed to equilibrate beforeeach data point was recorded.

[0270] Data Discussion:

[0271] Nucleic acid Molecular Beacons which form a hairpin structure areexpected to exhibit an increase in fluorescent intensity when heatedwhich is consistent with the melting of the hairpin stem and thephysical transition of the probe stem from a helix to a random coil.Consistent with any nucleic acid melting event, the process is expectedto be reversible thereby resulting in a decrease in fluorescence uponcooling of the sample caused by the resulting reformation of the helicalstructure. The expected melting phenomenon is documented for nucleicacid Molecular Beacons described by Tyagi et al. (See: Tyagi et al.Nature Biotechnology, 14: 303-308 (1996) at FIG. 3).

[0272] The results of the fluorescent thermal melting analysis of thePNA Molecular Beacons are summarized in the data presented in Table 8and presented graphically in FIGS. 7A, 7B1, 7B2, 7B3 and 7C. Withreference to Table 8, there are three different general Thermal Profilesobserved for the different constructs and under the conditions examined.These are represented in Table 8 as Types A, B and C.

[0273] Fluorescent Thermal Profile Type A is characterized by a anincrease in fluorescence intensity upon heating (melting) and acorrelating decrease in fluorescence intensity upon cooling(reannealing). These results are similar to those published for nucleicacid Molecular Beacons which form a loop and hairpin stem structure.Thus, a Type A Fluorescent Thermal Profile is consistent with theformation of a stable hairpin stem and loop structure. This phenomenonis, therefore, believed to be caused by the melting and reannealing of astem and loop structure in the PNA Molecular Beacon. However, applicantsonly claim that a Type A Fluorescent Thermal Profile is indicative offairly reversible fluorescence quenching, as other structures may beresponsible for or contribute to the observed phenomenon. TABLE 8Summary of Data Compiled In Experiments 20-22 Fluorescent HybridizationThermal Profile Profile Thermal Profile Probe No. CODE Observed ObservedObserved N-terminal Arm Forming Segments .001 5205 A A Sig, 6% .007 5105A A Sig, 7% .010 5005 B A  Sig, 19% .002 3203 A A Sig, 8% .008 3103 B ASig, 8% .009 4004 B A Sig, 8% C-terminal Arm Forming Segments .018 7027B A, B Sig. 6% .011A 5025 B A N. Sig, 5%  .006 3023 C C N. Sig, 8% Probing Sequence External To Arm Segments .017 5115 B B N. Sig, 14% .0053113 C C N. Sig, 10% Control Probes: No Arm Forming Segments .003 0000 BB No Data .004 0110 B B N. Sig, 5% 

[0274] Representatives of Type A Fluorescent Thermal Profiles areillustrated in FIG. 7A. The data presented in the Figure is for the PNAoligomers 0.001, 0.007 and 0.002. Data for both the melting (opencharacter) and the reannealing (solid character) is presented. Thesigmoidal transitions are consistent with a melting a reannealing of aduplex.

[0275] Fluorescent Thermal Profile Type B is characterized by anincrease in fluorescence intensity upon heating (melting), but, nosubstantial correlating decrease in fluorescence intensity upon coolingof the sample. Thus, under the conditions examined, the interactionswhich initially cause the quenching of fluorescence do not appear to bereadily reversible. Consequently, the data suggests that a PNA oligomerexhibiting a Type B Fluorescent Thermal Profile, does not exhibit allthe features of a True Molecular Beacon. Nonetheless, as seen by thehybridization assay data, a Type B Fluorescent Thermal Profile does notprohibit the PNA oligomer from functioning as a PNA Beacon.

[0276] Representatives of Type B Fluorescent Thermal Profiles areillustrated in FIGS. 8B1, 8B2 and 8B3. The data is presented in threesets so that each trace may be more clearly viewed. The data presentedin the Figures are for the PNA oligomers 0.10, 0.008, 0.009 (FIG. 7B1),0.018, 0.011A, 0.017, (FIGS. 7B2), and 0.003 and 0.004, (FIG. 7B3). Datafor both the melting (open character) and the reannealing (solidcharacter) is presented.

[0277] Fluorescent Thermal Profile Type C is characterized by a highinitial fluorescent intensity which initially decreases with heating andagain decreases even further upon cooling of the sample. The highinitial fluorescent intensity suggests that this construct does notexhibit the initial fluorescence quenching observed with most of theother PNA constructs examined. The constant decrease in fluorescentintensity upon cooling is not well understood. Nevertheless, as seen bythe hybridization assay data, a Type C, Fluorescent Thermal Profile doesnot prohibit the PNA oligomer from functioning as a PNA Beacon.

[0278] Representatives of Type C Fluorescent Thermal Profiles areillustrated in FIG. 7C. The data presented in the FIG. 7C is for the PNAoligomers 0.005 and 0.006. Data for both the melting (open character)and the reannealing (solid character) is presented.

Example 21

[0279] Analysis of Hybridization Assay Data

[0280] General Experimental Procedures:

[0281] All hybridization assay data was collected using a Wallac 1420VICTOR equipped with a F485 CW-lamp filter and a F535 Emission filter.The NUNC MaxiSorp, breakapart microtitre plate was used as the reactionvessel. Each microtitre plate was prewashed with Hyb. Buffer at roomtemperature for 15 minutes before the reaction components were added.Total reaction volume was 100 μL in Hyb. Buffer.

[0282] Stock solution of purified PNA probe was prepared by dissolvingthe PNA in 50% aqueous N,N′-dimethylformamide (DMF). From this PNA Stockwas prepared a solution of each PNA at a concentration of 25 pmole/μL byserial dilution of the PNA Stock with 50% aqueous DMF.

[0283] Stock solution of purified wt k-ras DNA was prepared bydissolving the purified DNA in TE (10 mM Tris. HCl pH 8.0; 1.0 mM EDTA,Sigma Chemical). From this DNA Stock was prepared a solution of wt k-rasDNA at a concentration of 100 pmol/99 μL by serial dilution of the DNAStock with Hyb. Buffer.

[0284] Each reaction sample used for analysis was prepared by combining1 μL of the appropriate PNA oligomer (25 pmole/μL) with either of 99 μLof wt k-ras DNA stock or 99 μL of Hyb. Buffer (control) as needed toprepare 100 μL of sample.

[0285] Samples were mixed and then fluorescence intensity monitored withtime using the Wallac VICTOR instrument. Samples were run in triplicateto insure reproducible results. Data was acquired for 20-25 minutesafter the reactants were mixed and then the wells were sealed and theplate heated to 42-50° C. in an incubator for 30-40 minutes. Aftercooling to ambient temperature, the wells were unsealed and then another10 data points were collected over approximately five minutes.

[0286] Data Discussion:

[0287] Nucleic acid Molecular Beacons which form a hairpin stem and loopstructure are expected to exhibit an increase in fluorescent intensityupon hybridization of the probing sequence to complementary nucleicacid. The expected increase in fluorescent intensity is documented forDNA Molecular Beacons described by Tyagi et al. (See: Tyagi et al.Nature Biotechnology, 14: 303-308 (1996)).

[0288] The results of the hybridization analysis of the PNA oligomersare summarized in Table 8 and presented graphically in FIGS. 8A1, 8A2,8A3, 8B and 8C. With reference to Table 8, there are three differentgeneral Hybridization Profiles observed for the different constructsexamined. These are represented in Table 8 as Types A, B and C. In FIG.10, the signal to noise ratio (before and after heating) for all probesexamined are graphically illustrated.

[0289] Hybridization Profile Type A is characterized by the increase influorescence intensity in samples containing complementary target DNA ascompared with samples containing only the PNA oligomer. After heating,the fluorescent intensity of samples containing target sequenceincreases but the background fluorescence of the control sample(s) doesnot significantly change. Because the PNAs possess a very low inherentfluorescence, the probes which exhibit a Type A, Hybridization Profilegenerally have the highest signal to noise ratios. Representatives ofType A Hybridization Profiles are illustrated in FIGS. 8A1, 8A2 and 8A3.The data is presented in two separate graphical illustrations to clarifythe presentation. The data presented in the Figures is for the PNAoligomers 0.001, 0.007, 0.010 (FIG. 8A1), and 0.002, 0.008, 0.009 (FIG.8A2), and 0.11A, 0.017 and 0.018 (FIG. 8A3).

[0290] Hybridization Profile Type B is characterized by the very rapidincrease in fluorescence intensity in samples containing complementarytarget DNA as compared with samples containing only the PNA oligomer.The fluorescence intensity quickly reaches a plateau which does notsignificantly change (if at all) after heating. The backgroundfluorescence of the control sample(s) does not change significantly evenafter heating. This suggest that the hybridization event rapidly, andwith little resistance, reaches a binding equilibrium without anyrequirement for heating. Representatives of Type B HybridizationProfiles are illustrated in FIG. 8B. The data presented in FIG. 8B isfor the PNA oligomers 0.018, 0.003 and 0.004 though PNA oligomer 0.018does not exhibit all the characteristics of a Type B HybridizationProfile. Specifically, the signal for probe 0.018 does not appear toincrease after heating (Type B) but the hybridization kinetics appear tobe more like a Type A Hybridization Profile.

[0291] Control probes 0.003 and 0.004 (herein referred to as PNAMolecular Beacons) exhibit a Type B Hybridization Profile. Thus, therapid hybridization kinetics of the Type B Hybridization Profile isprobably the result of having no stable hairpin stem, or any otherstrong force, which can stabilize the non fluorescent polymer form.Nonetheless, the dynamic range (signal to noise ratio) observed in thehybridization assay of these probes is typically quite high and suggeststhat forces other than the hydrogen bonding of complementary nucleobasesof arm segments can stabilize the interactions between the donor andacceptor moieties. The data presented in Example 19 suggests thatlabel/label interactions can be quite strong and may be an importantfactor in this surprising result.

[0292] Though the background (noise) is higher for the 0.003 and 0.004probes, as compared with the 0.001, 0.002, 0.007, 0.009 and 0.010probes, the fluorescence intensity after hybridization is higher thanthat observed in any probes yet examined. As a result of the higherbackground, PNA oligomers 0.003 and 0.004 have a very favorable signalto noise ratio. This S/N ratio is nearly as favorable as any (and betterthan some) of the other PNA oligomers examined whether or not theypossess arm segments. The data demonstrates that it is not necessary tohave arm forming segments to create a probe which exhibits an initiallow fluorescence intensity and a corresponding increase in fluorescencesignal upon the binding of the probe to a target sequence.

[0293] Hybridization Profile C is characterized by a moderate increasein fluorescence intensity in samples containing target DNA as comparedwith samples containing only the PNA oligomer. The fluorescenceintensity quickly reaches a plateau which does not significantly change(if at all) after heating. The background fluorescence of the controlsample(s) is relatively high but does not change significantly evenafter heating. Hybridization Profiles B and C differ primarily becausethe background fluorescence in the control samples, containing no targetnucleic acid, are dramatically higher in Hybridization Profile Type C.The hybridization data obtained for samples containing complementarynucleic acid, suggests that the hybridization event rapidly, and withlittle resistance, reaches equilibrium. However, the very highbackground signal suggests that the forces which should hold the donorand acceptor moieties in close proximity are not strong enough in theseconstructs to effectively quench the fluorescent signal. As aconsequence of the moderate increase in fluorescence upon binding to thetarget sequence and the higher than usual intrinsic fluorescence a PNAMolecular Beacon, which exhibits a Type C Hybridization Profile, has avery low signal to noise ratio. Representatives of Type C HybridizationProfiles are illustrated in FIG. 8C. The data presented in FIG. 8C isfor the PNA oligomers 0.006 and 0.005, respectively.

Example 22

[0294] Ultraviolet Thermal Profile Analysis

[0295] The data collected for this Example was intended to determinewhether the fluorescence vs. temperature analysis presented in Example20 correlated with ultraviolet (UV) absorbance (260 nm) vs. temperatureplots. Additionally, concentration dependency of the traces was alsoexamined in order to determine whether the PNA Molecular Beacons listedin Table 1 (except probe 0.003) existed as a hairpin or as a multimer.

[0296] Materials and Methods:

[0297] The purified probes were dissolved in the Hyb. Buffer to aconcentration which was intended to be approximately 5-7.5 μM. Howeverit was determined that the PNA Molecular Beacons were too insoluble suchthat a large proportion of the PNA probe existed in a suspension. Thesolutions were centrifuged to remove suspended matter and therefore themost concentrated samples examined were estimated to have aconcentration of approximately 2.5 μM or less. The most concentratedstocks were then serially diluted with Hyb. Buffer two times so that foreach sample, a total of three concentrations could be examined. Allsamples were analyzed using a Cary 100 Bio UV-Visible Spectrophotometer(Varian Instruments) equipped with a 6×6 thermostatable multicell blockrunning Win UV Bio application software package. To a 10×10 UV cell(Starna Cells, P/N 21-Q-10) was added a 7.2 mm stir bar and the 2.5 mLof each sample of the dilution series. The stirring accessory was usedduring all analysis. All samples were thermally denatured and reannealedprior to data collection by having the instrument rapidly ramp to atleast 90C. and then holding for 5 minutes before returning to thestarting temperature of 20° C. After the premelt, it was preferable toallow the samples to remain at the starting temperature for at least 30minutes to reach equilibrium before beginning data collection. Data forboth dissociation and reannealing was collected and analyzed. Thetemperature range over which data was collected was 20-90° C. Thetemperature ramp rate for both dissociation and reannealing was 0.5°C./min. The absorbance (260 nm, averaged over a 2-3 second collection)was plotted vs. the temperature of the multicell block.

[0298] Results:

[0299] Factors to be considered in analyzing the absorbance vs.temperature plots include whether the transition was sigmoidal, whetherand to what extent there was any hysteresis and the percenthyperchromicity (for the purposes of this discussion the percenthyperchromicity will be defined as the approximate percent differencebetween the absorbance at 20° C. and the absorbance at 90° C.). Summaryof the data obtained by analysis of the absorbance vs. temperature plotsis presented in Table 8.

[0300] Probes 0.001, 0.002, 0.007, 0.008, 0.009, 0.010 and 0.018exhibited a sigmoidal transition as indicated by “Sig” in Table 8. Thesigmoidal transition is characteristic of the melting and reannealing ofa duplex or hairpin. Probes. 0.004, 0.005, 0.006, 011A and 0.017 allexhibited a non-sigmoidal transition as indicated by “N. Sig” in Table8. Curiously, the shape of the non-sigmoidal transition was essentiallythe same in all cases except for 0.011A. For these probes the increasein absorbance as a function of temperature appeared to be almost linear.The non-sigmoidal shape of these curves suggest that the transition isnot the result of the melting and reannealing of a duplex structure.Thus, these probe are not likely to exist as hairpins or multimers.

[0301] Not a single probe examined was without a noticeable hysteresis.Though the extent of hysteresis varied widely, the presence of aconspicuous hysteresis indicates that the probes are resistant toreturning to their original confirmation as a hairpin, mulitmer or otherconfirmation. Though this result was generally observed in thefluorescence vs. temperature plots, the absorbance traces were far morereversible upon cooling as compared with the data observed in thefluorescence vs. temperature plots. Therefore, the substantialdifferences between the fluorescence vs. temperature and absorbance vs.temperature plots are not understood. Moreover, it is unclear why onlyprobes 0.001, 0.002 and 0.007 exhibited a reversible decrease influorescence upon cooling whereas other probes did not. However, thedata suggests that the longer flexible linkages and longer arm segmentspromote favorable properties since probes 0.001 and 0.002 both possess 2flexible linkages and probe 0.007, though is possesses only one flexiblelinkage, it comprises 5 subunit arm segments.

[0302] For comparison, normalized absorbance vs. temperature andfluorescence vs. temperature data for probe 0.001 was overlaid since theplots looked relatively similar. The overlaid data is presented in FIG.9. With reference to FIG. 9, the absorbance vs. temperature andfluorescence vs. temperature data is fairly superimposible. Theabsorbance vs. temperature and fluorescence vs. temperature plots forprobe 0.002 and 0.007 were likewise very similar thought the data hasnot yet been overlaid. The good correlation between the absorbance vs.temperature and fluorescence vs. temperature plots suggests that thesame transition is being measured in both analyses and it is likely tobe a helix to coil transition.

[0303] Except for probes 0.010, 0.005 and 0.017, the percenthyperchromicity is less than 10 percent. Generally, the hyperchromiceffect for a duplex to random coil transition is greater than 10percent. The hyperchromicity for the DNA and PNA probes examined inExample 19 were all better than 15 percent. Thus, the lower thanexpected hyperchromic effect for substantially all probes, except probe,0.010 which exhibits a 19 percent hyperchromic effect, is not wellunderstood. Nevertheless, the values are not consistent with at meltingof a duplex of a hairpin even for probes which exhibited a sigmoidaltransition.

[0304] Finally, the data obtained by applicants was inconclusive withregard to whether the PNA Molecular Beacons listed in Table 1 exist as ahairpin or multimer because the scatter in the data at the lowerconcentrations made it impossible to obtain reliable derivativeinformation from which the Tm values are determined. Though the datagenerated in Example 19 would suggest that probes comprising armsegments of six or less subunits are not likely to form hairpins, theeffect of flexible linkers was not fully evaluated in that Example.Thus, it remains unknown whether any of the probes listed in Table 1exist primarily as hairpins.

[0305] In summary, the data presented in this Example 22 is inconclusiveas to whether the PNA Molecular Beacons listed in the Table existprimarily as hairpins or mulitmers. It does however show there is goodagreement between the absorbance vs. temperature plots and thefluorescence vs. temperature plots for probes 0.001, 0.002 and 0.007.The lack of correlation between absorbance vs. temperature plots and thefluorescence vs. temperature plots for other probes supports the theorythat the probes may adopt structures other than hairpins or multimers.This theory is supported by the non-sigmoidal transitions, thesubstantial hysteresis and the very low percent hyperchromicity for mostof the probes.

[0306] General Discussion of the Data Presented in Examples 19-22

[0307] Though all the probes examined exhibited a detectable increase influorescent signal in the presence of a target sequence, the probeswhich exhibit properties which are most like nucleic acid MolecularBeacons are probes 0.001, 0.002 and 0.007. These probes process veryfavorable signal to noise ratios, exhibit sigmoidal transitions duringmelting and also readily reannealed upon cooling whether the analysiswas by fluorescence or absorbance. This data indicates that probes ofthis configuration form duplexes which dissociate upon hybridization orthermal melting to produce an increase in detectable signal though it isnot known whether or not these probes exist primarily as hairpins.Whether hairpins or not, the favorable characteristics of these probescorrelate with the presence of flexible linkages and arm segments in therange of 3 to 5 subunits in length. Furthermore, the data in Example 19in combination with the data for probe 0.018, in Examples 20-22,demonstrate that long arm segments of 7 to 9 subunits substantiallyreduce signal thereby resulting in very poor signal to noise ratios.Consequently, long arm segments are a disfavored configuration for a PNAMolecular Beacon.

[0308] Though probes 0.001, 0.002 and 0.007 exhibited the most favorableproperties, all the probes listed in Table 1, except for control probes0.003 and 0.004, are PNA Molecular Beacons because they comprise armsegments and appropriate labeling and also exhibit a detectable changein a property of a label which correlates with the binding of the probeto a target sequence. The nature of the forces which result influorescence quenching of the other PNA probes is not well understood,though it is likely that nucleobase-nucleobase, electrostatic andhydrophobic-hydrophobic interactions contribute to fix the probes in afavorable secondary structure until this is altered by hybridization.

[0309] Surprisingly, the control probes 0.003 and 0.004, which have noarm forming segments, exhibit a correlation between increasedfluorescence intensity and binding of the probe to target sequence.Remarkably these probe exhibit a very good signal to noise ratio inhybridization assays. Thus, it has been demonstrated that PNA oligomersneed not comprise regions of complementary nucleobases which are, bydesign, intended to form a hairpin to thereby exhibit many of thefavorable characteristics of a nucleic acid Molecular Beacon. Since PNAoligomers 0.003 and 0.004 cannot form duplexes, this result demonstratesthat other types of secondary structures can result in fluorescencequenching until the probe hybridizes to a target sequence.

EQUIVALENTS

[0310] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention as defined by the appended claims. Those skilled in theart will be able to ascertain, using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed in the scope of the claims.

1 10 1 31 DNA Artificial Sequence misc_feature (1) 5′ Biotin 1gtggtagttg gagctggtgg cgtaggcaag a 31 2 27 DNA Artificial SequenceDescription of Artificial Sequence SYNTHETIC PROBE OR TARGET 2ggtagtgtct ggtgatgctg gaggcaa 27 3 11 DNA Artificial Sequencemisc_feature (1) 5′Fluorescein 3 gccaccagct c 11 4 13 DNA ArtificialSequence misc_feature (1) 5′ Fluorescein 4 cgccaccagc tcc 13 5 15 DNAArtificial Sequence misc_feature (1) 5′ Fluorescein 5 acgccaccag ctcca15 6 17 DNA Artificial Sequence misc_feature (1) 5′ Fluorescein 6tacgccacca gctccaa 17 7 20 DNA Artificial Sequence Description ofArtificial Sequence SYNTHETIC PROBE OR TARGET 7 atgactgaat ataaacttgt 208 20 DNA Artificial Sequence Description of Artificial SequenceSYNTHETIC PROBE OR TARGET 8 ctctattgtt ggatcatatt 20 9 111 DNA Homosapiens Description of Artificial Sequence SYNTHETIC PROBE OR TARGET 9gagataacaa cctagtataa gcaggtgttt tactaagact taatcgactt agcagttccg 60tgagaacgga tgcggtgttc gaggttgatg gtgttcaaat ataagtcagt a 111 10 111 DNAHomo sapiens Description of Artificial Sequence SYNTHETIC PROBE ORTARGET 10 ctctattgtt ggatcatatt cgtccacaaa atgattctga attagctgtatcgtcaaggc 60 actcttgcct acgccacaag ctccaactac cacaagttta tattcagtca t111

We claim:
 1. A method for in-situ analysis of a target sequence of anorganism of interest in a sample, said method comprising: a) providing asample of fixed cells for in-situ analysis; b) treating the sample witha polymer of covalently linked subunits, said polymer comprising; i) anon-polynucleotide probing nucleobase sequence that is complementary orsubstantially complementary to a target sequence; ii) a first armsegment and a second arm segment that are covalently linked to, orembedded within, the probing nucleobase sequence and wherein at leastone of the first or second arm segments is covalently linked to theprobing nucleobase sequence through a flexible linkage; iii) at leastone linked donor moiety and at least one linked acceptor moiety, whereinsaid donor and acceptor moieties are covalently linked to the polymer atpositions that are separated by at least a portion of the probingnucleobase sequence; c) detecting, identifying or quantitating thehybridization of the polymer to the target sequence, under suitablein-situ hybridization conditions, wherein the presence, absence oramount of target sequence present in the sample is correlated with achange in detectable signal associated with at least one donor oracceptor moiety of the polymer; and d) detecting, identifying orquantitating the organism of interest in the sample based upon thepresence, absence or amount of the hybridization of the polymer to thetarget sequence that is determined.
 2. The method of claim 1, whereinexcess polymer is not washed away prior to performing step (c).
 3. Themethod of claim 1, wherein blocker probes are added to improve assayperformance.
 4. The method of claim 1, wherein the determination ofdetectable signal is made using a microscope, laser scanning device orflow cytometer.
 5. The method of claim 1, wherein the organism ofinterest is selected from the group consisting of a bacteria or a virus.6. The method of claim 1, wherein the method is repeatedly performed todetermine the effect of antimicrobial agents on the growth of one ormore microorganisms.
 7. The method of claim 1, wherein the method isselected to determine target sequences that are particular to organismsfound in food, beverages, water, pharmaceutical products, personal careproducts, dairy products or environmental samples.